A theoretical study on the effect of surface roughness on mass transport and transformation in biofilms

This modeling study evaluates the influence of biofilm geometrical characteristics on substrate mass transfer and conversion rates. A spatially two-dimensional model was used to compute laminar fluid flow, substrate mass transport, and conversion in irregularly shaped biofilms. The flow velocity above the biofilm surface was varied over 3 orders of magnitude. Numerical results show that increased biofilm roughness does not necessarily lead to an enhancement of either conversion rates or external mass transfer. The average mass transfer coefficient and Sherwood numbers were found to decrease almost linearly with biofilm area enlargement in the flow regime tested. The influence of flow, biofilm geometry and biofilm activity on external mass transfer could be quantified by Sh-Re correlations. The effect of biofilm surface roughness was incorporated in this correlation via area enlargement. Conversion rates could be best correlated to biofilm compactness. The more compact the biofilm, the higher the global conversion rate of substrate. Although an increase of bulk fluid velocity showed a large effect on mass transfer coefficients, the global substrate conversion rate per carrier area was less affected. If only diffusion occurs in pores and channels, then rough biofilms behave as if they were compact but having less biomass activity. In spite of the fact that the real biofilm area is increased due to roughness, the effective mass transfer area is actually decreased because only biofilm peaks receive substrate. This can be explained by the fact that in the absence of normal convection in the biofilm valleys, the substrate gradients are still largely perpendicular to the carrier. Even in the cases where convective transport dominates the external mass transfer process, roughness could lead to decreased conversion rates. The results of this study clearly indicate that only evaluation of overall conversion rates or mass fluxes can describe the correct biofilm conversion, whereas interpretation of local concentration or flow measurements as such might easily lead to erroneous conclusions.     

Influence of biomass production and detachment forces on biofilm structures in a biofilm airlift suspension reactor

The influence of process conditions (substrate loading rate and detachment force) on the structure of biofilms grown on basalt particles in a Biofilm Airlift Suspension (BAS) reactor was studied. The structure of the biofilms (density, surface shape, and thickness) and microbial characteristics (biomass yield) were investigated at substrate loading rates of 5, 10, 15, and 20 kg COD/m3. day with basalt concentrations of 60 g/L, 150 g/L, and 250 g/L. The basalt concentration determines the number of biofilm particles in steady state, which is the main determining factor for the biofilm detachment in these systems. In total, 12 experimental runs were performed. A high biofilm density (up to 67 g/L) and a high biomass concentration was observed at high detachment forces. The higher biomass content is associated with a lower biomass substrate loading rate and therefore with a lower biomass yield (from 0.4 down to 0.12 gbiomass/gacetate). Contrary to general beliefs, the observed biomass detachment decreased with increasing detachment force. In addition, smoother (fewer protuberances), denser and thinner compact biofilms were obtained when the biomass surface production rate decreased and/or the detachment force increased. These observations confirmed a hypothesis, postulated earlier by Van Loosdrecht et al. (1995b), that the balance between biofilm substrate surface loading (proportional to biomass surface production rate, when biomass yield is constant) and detachment force determines the biofilm structure. When detachment forces are relatively high only a patchy biofilm will develop, whereas at low detachment forces, the biofilm becomes highly heterogeneous with many pores and protuberances. With the right balance, smooth, dense and stable biofilms can be obtained. Copyright 1998 John Wiley & Sons, Inc.     

Three-dimensional biofilm structure quantification

Quantitative parameters describing biofilm physical structure have been extracted from three-dimensional confocal laser scanning microscopy images and used to compare biofilm structures, monitor biofilm development, and quantify environmental factors affecting biofilm structure. Researchers have previously used biovolume, volume to surface ratio, roughness coefficient, and mean and maximum thicknesses to compare biofilm structures. The selection of these parameters is dependent on the availability of software to perform calculations. We believe it is necessary to develop more comprehensive parameters to describe heterogeneous biofilm morphology in three dimensions. This research presents parameters describing three-dimensional biofilm heterogeneity, size, and morphology of biomass calculated from confocal laser scanning microscopy images. This study extends previous work which extracted quantitative parameters regarding morphological features from two-dimensional biofilm images to three-dimensional biofilm images. We describe two types of parameters: (1) textural parameters showing microscale heterogeneity of biofilms and (2) volumetric parameters describing size and morphology of biomass. The three-dimensional features presented are average (ADD) and maximum diffusion distances (MDD), fractal dimension, average run lengths (in X, Y and Z directions), aspect ratio, textural entropy, energy and homogeneity. We discuss the meaning of each parameter and present the calculations in detail. The developed algorithms, including automatic thresholding, are implemented in software as MATLAB programs which will be available at site prior to publication of the paper.     

Two-dimensional model of biofilm detachment caused by internal stress from liquid flow

A two-dimensional model for biofilm growth and detachment was used to evaluate the effect of detachment on biofilm structures. The detachment process is considered to be due to internal stress created by moving liquid past the biofilm. This model generated a variety of realistic biofilm-formation patterns. It was possible to model in a unified way two different biofilm detachment processes, erosion (small-particle loss), and sloughing (large-biomass-particle removal). The distribution of the fraction from total biomass detached as a function of detached particle mass, gives indications about which of the two mechanisms is dominant. Model simulations indicate that erosion makes the biofilm surface smoother. Sloughing, in contrast, leads to an increased biofilm-surface roughness. Faster growing biofilms have a faster detachment rate than slow-growing biofilms, under similar hydrodynamic conditions and biofilm strength. This is in perfect accordance with the experimental evidence showing that detachment is dependent on both shear- and microbial-growth rates. High growth rates trigger instability in biofilm accumulation and abrupt biomass loss (sloughing). Massive sloughing can be avoided by high liquid shear, combined with low biomass growth rates. As the modeling results show, the causes for sloughing must be sought not only in the biofilm strength, but also in its shape. Several "mushroom-like" biofilm structures like those repeatedly reported in the literature occurred, due to a combined effect of nutrient depletion and breaking at the colony base. A rough carrier surface promotes biofilm development in hydrodynamic conditions in which the biofilm on a flat surface would not form. Although biofilm patches filled completely the cavity in which they started to grow, they were unable to spill over the carrier peaks and to fully colonize the substratum.     

Effect of diffusive and convective substrate transport on biofilm structure formation: a two-dimensional modeling study

A two-dimensional model for quantitative evaluation of the effect of convective and diffusive substrate transport on biofilm heterogeneity was developed. The model includes flow computation around the irregular biofilm surface, substrate mass transfer by convection and diffusion, biomass growth, and biomass spreading. It was found that in the absence of detachment, biofilm heterogeneity is mainly determined by internal mass transfer rate of substrates and by the initial percentage of carrier-surface colonization. Model predictions show that biofilm structures with highly irregular surface develop in the mass transfer-limited regime. As the nutrient availability increases, there is a gradual shift toward compact and smooth biofilms. A smaller fraction of colonized carrier surface leads to a patchy biofilm. Biofilm surface irregularity and deep vertical channels are, in this case, caused by the inability of the colonies to spread over the whole substratum surface. The maximum substrate flux to the biofilm was greatly influenced by both internal and external mass transfer rates, but not affected by the inoculation density. In general, results of the present model were similar to those obtained by a simple diffusion-reaction-growth model.     

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.     

Optical method for long-term and large-scale monitoring of spatial biofilm development

A method was developed that allows biofilm monitoring on the square centimeter scale over extended periods of time. The method is based on image acquisition using a desktop scanner and subsequent image analysis. It was shown that results from grey level analysis are highly correlated with physical properties of the biofilm like average biomass and biofilm thickness. The scanner method was applied to monitor overall biofilm growth, detachment, and surface roughness during two 3 and 4 week long experiments. Two significantly different growth dynamics during the biofilm development could be identified, depending on the biofilm history. Surface roughness on transects in flow direction was always higher than on transects perpendicular to the flow, reflecting the anisotropic characteristics of biofilms growing in a flow field.     

Abrasion of suspended biofilm pellets in airlift reactors: importance of shape, structure, and particle concentrations

The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. (c) 1997 John Wiley & Sons, Inc.     

Development of pure culture biofilms of P. putida on solid supports

Pseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined.Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.     

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Measuring local flow velocities and biofilm structure in biofilm systems with magnetic resonance imaging (MRI)

The characterization of substrate transport in the bulk phase and in the biofilm matrix is one of the problems which has to be solved for the verification of biofilm models. Additionally, the surface structure of biofilms has to be described with appropriate parameters. Magnetic resonance imaging (MRI) is one of the promising methods for the investigation of transport phenomena and structure in biofilm systems. The MRI technique allows the noninvasive determination of flow velocities and biofilm structures with a high resolution on the sub-millimeter scale. The presented investigations were carried out for defined heterotrophic biofilms which were cultivated in a tube reactor at a Reynolds number of 2000 and 8000 and a substrate load of 6 and 4 g/m2d glucose. Magnetic resonance imaging provides both structure data of the biofilm surface and flow velocities in the bulk phase and at the bulk/biofilm interface. It is shown that the surface roughness of the biofilms can be determined in one experiment for the complete cross section of the test tubes both under flow and stagnant conditions. Furthermore, the local shear stress was calculated from the measured velocity profiles. In the investigated biofilm systems the local shear stress at the biofilm surface was up to 3 times higher compared to the mean wall shear stress calculated on the base of the mean flow velocity.     

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.     

Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start-up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start-up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start-up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.The Pirt-maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 +/- 0.08 C-mol/C-mol, the maintenance value 0.019 +/- 0.012 C-mol/(C-mol h). The lowest actual biomass yield measured in this study was 0.15 C-mol/C-mol. (c) 1994 John Wiley & Sons, Inc.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Processes governing primary biofilm formation

Biofilm accumulation under turbulent flow condition on the surface of a circular tube is the net result of several process including the following: (1) transport and firm adhesion of soluble components and microbial cell to the surface; (2) metabolic conversions within the biofilm in cluding growth and maintenance decay process; (3) detachment of portions of the biofilm and reentrainment in the bulk fluid. Experiments in tabular reactor were designed to measure the rates of these process during the early stages of biofilm accumulation as a function of the Reynolds number and suspended biomass concentration. Results indicate deposition (i.e., combined transport and adsorption) is only important in the very early stages of biofilm accumulation and is significantly influenced by negligible for the thin biofilms encountered in these experiments. Net biofilm production rates in all experiments decrease to same level and this level is not affected by changes in Reynolds number or suspended biomass concentration. Biofilm detachment rate increases continuously with biofilm accumulation and with increasing Reynolds number.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.