The effect of biofilm permeability on bio-clogging of porous media

A 3D Biofilm model, appropriate for complex porous media support structures, is successfully modified such that non‐zero permeability of biofilms structures is enabled. A systematic study is then conducted into the influence of biofilm permeability on overall biomass growth rate. This reveals a significant influence at large biofilm concentrations; even when the permeability of the biomass is 1.25% of that of the free pore space, biomass accumulation increased by a factor of ～3 over 40 h. The effect is shown to be retained when allowing for biomass detachment or erosion as a consequence of adjacent velocity shear. We conclude that biofilm permeability should be included in biofilm models and that further experimental work is required to better describe the link between biofilm permeability and local microstructure. Biotechnol. Bioeng. 2012; 109:1031–1042. © 2011 Wiley Periodicals, Inc.     

Pore‐network modeling of biofilm evolution in porous media

The influence of bacterial biomass on hydraulic properties of porous media (bioclogging) has been explored as a viable means for optimizing subsurface bioremediation and microbial enhanced oil recovery. In this study, we present a pore network simulator for modeling biofilm evolution in porous media including hydrodynamics and nutrient transport based on coupling of advection transport with Fickian diffusion and a reaction term to account for nutrient consumption. Biofilm has non‐zero permeability permitting liquid flow and transport through the biofilm itself. To handle simultaneous mass transfer in both liquid and biofilm in a pore element, a dual‐diffusion mass transfer model is introduced. The influence of nutrient limitation on predicted results is explored. Nutrient concentration in the network is affected by diffusion coefficient for nutrient transfer across biofilm (compared to water/water diffusion coefficient) under advection dominated transport, represented by mass transport Péclet number >1. The model correctly predicts a dependence of rate of biomass accumulation on inlet concentration. Poor network connectivity shows a significantly large reduction of permeability, for a small biomass pore volume. Biotechnol. Bioeng. 2011;108: 2413–2423. © 2011 Wiley Periodicals, Inc.     

Biomass evolution in porous media and its effects on permeability under starvation conditions

The purpose of this study was to understand bacteria profile modification and its applications in subsurface biological operations such as biobarrier formation, in situ bioremediation, and microbial-enhanced oil recovery. Biomass accumulation and evolution in porous media were investigated both experimentally and theoretically. To study both nutrient-rich and carbon-source-depleted conditions, Leuconostoc mesenteroides was chosen because of its rapid growth rate and exopolymer production rate. Porous micromodels were used to study the effects of biomass evolution on the permeability of a porous medium. Bacterial starvation was initiated by switching the feed from a nutrient solution to a buffer solution in order to examine biofilm stability under nutrient-poor conditions. Four different evolution patterns were identified during the nutrient-rich and nutrient-depleted conditions used in the micromodel experiments. In phase I, the permeability of the porous micromodel decreased as a result of biomass accumulation in pore bodies and pore throats. In phase II, starvation conditions were initiated. The depletion of nutrient in the phase II resulted in slower growth of the biofilm causing the permeability to reach a minimum as all the remaining nutrients were consumed. In phase III, permeability began to increase due to biofilm sloughing caused by shear stress. In phase IV, shear stress remained below the critical shear stress for sloughing and the biofilm remained stable for long periods of time during starvation. The critical shear stress for biofilm sloughing provided an indication of biofilm strength. Shear removal of biofilms occurred when shear stress exceeded critical shear stress. A network model was used to describe the biofilm formation phenomenon and the existence of a critical shear stress. Simulations were in qualitative agreement with the experimental results, and demonstrate the existence of a critical shear stress.     

Relationship between glycocalyx and povidone-iodine resistance in Pseudomonas aeruginosa (ATCC 27853) biofilms.

Biofilm-embedded bacteria are generally more resistant to antimicrobial agents than are planktonic bacteria. Two possible mechanisms for biofilm resistance are that the glycocalyx matrix secreted by cells in a biofilm reacts with and neutralizes the antimicrobial agent and that the matrix creates a diffusion barrier to the antimicrobial agent. This study was therefore conducted to examine the relationship between glycocalyx and enhanced povidone-iodine resistance in biofilms of Pseudomonas aeruginosa (ATCC 27853). Biofilms were generated by inoculation of polycarbonate membranes with broth-grown cells and incubation of them on the surfaces of nutrient agar plates. The quantities of glycocalyx material per cell were found not to be significantly different between biofilm and planktonic samples. Transmission electron microscopy showed that the distributions of glycocalyx material around cells differed in biofilm and in planktonic samples. Addition of alginic acid to planktonic cell suspensions resulted in a slight increase in resistance to povidone-iodine, suggesting some neutralizing interaction. However, the iodine demands created by biofilm and planktonic samples of equivalent biomass were not significantly different and, therefore, do not explain the contrast in resistance observed between biofilm and planktonic samples. Examination of the relationship between cell death and biomass detachment from the glycocalyx matrix revealed that most cell death occurred in the fraction of biomass that detached from a biofilm during treatment. The overall rate of iodine diffusion through biofilms was not different from that of planktonic cells collected on a polycarbonate membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biofilm development and the dynamics of preferential flow paths in porous media

A two-dimensional pore-scale numerical model was developed to evaluate the dynamics of preferential flow paths in porous media caused by bioclogging. The liquid flow and solute transport through the pore network were coupled with a biofilm model including biomass attachment, growth, decay, lysis, and detachment. Blocking of all but one flow path was obtained under constant liquid inlet flow rate and biomass detachment caused by shear forces only. The stable flow path formed when biofilm detachment balances growth, even with biomass weakened by decay. However, shear forces combined with biomass lysis upon starvation could produce an intermittently shifting location of flow channels. Dynamic flow pathways may also occur when combined liquid shear and pressure forces act on the biofilm. In spite of repeated clogging and unclogging of interconnected pore spaces, the average permeability reached a quasi-constant value. Oscillations in the medium permeability were more pronounced for weaker biofilms.     

Experimental and theoretical investigations of rotating algae biofilm reactors (RABRs): Areal productivity,nutrient recovery,and energy efficiency

Microalgae biofilms have been demonstrated to recover nutrients from wastewater and serve as biomass feedstock for bioproducts. However, there is a need to develop a platform to quantitatively describe microalgae biofilm production, which can provide guidance and insights for improving biomass areal productivity and nutrient uptake efficiency. This paper proposes a unified experimental and theoretical framework to investigate algae biofilm growth on a rotating algae biofilm reactor (RABR). Experimental laboratory setups are used to conduct controlled experiments on testing environmental and operational factors for RABRs. We propose a differential–integral equation-based mathematical model for microalgae biofilm cultivation guided by laboratory experimental findings. The predictive mathematical model development is coordinated with laboratory experiments of biofilm areal productivity associated with ammonia and inorganic phosphorus uptake by RABRs. The unified experimental and theoretical tool is used to investigate the effects of RABR rotating velocity, duty cycle (DC), and light intensity on algae biofilm growth, areal productivity, nutrient uptake efficiency, and energy efficiency in wastewater treatment. Our framework indicates that maintaining a reasonable light intensity range improves biomass areal productivity and nutrient uptake efficiency. Our framework also indicates that faster RABR rotation benefits biomass areal productivity. However, maximizing the nutrient uptake efficiency requires a reasonably low RABR rotating speed. Energy efficiency is strongly correlated with RABR rotating speed and DC.     

A new approach to model the spatiotemporal development of biofilm phase in porous media

Bacteria can exist within biofilms that are attached to the solid matrix of a porous medium. Under certain conditions, the biomass can fully occupy the pore space leading to reduced hydraulic conductivity and mass transport. Here, by treating biofilm as a growing, high-viscosity phase, a novel macroscopic approach to model biofilm spatial expansion and its corresponding effects on porous medium hydraulic properties is presented. The separate yet coupled flow of the water and biofilm phases is handled by using relative permeability curves that allow for biofilm movement within the porous medium and bioclogging effects. Fluid flow is governed by Darcy's law and component transport is set by the convection-diffusion equation reaction terms for each component. Here, the system of governing equations is solved by using a commercial multiphase flow reservoir simulator, which is used to validate the model against published laboratory experiments. A comparison of the model and experimental observations reveal that the model provides a reasonable means to predict biomass development in the porous medium. The results reveal that coupled flow of water and movement of biofilm, as described by relative permeability curves, is complex and has a large impact on the development of biomass and consequent bioclogging in the porous medium.     

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.     

Modelling biofilm‐induced formation damage and biocide treatment in subsurface geosystems

Biofilm growth in subsurface porous media, and its treatment with biocides (antimicrobial agents), involves a complex interaction of biogeochemical processes which provide non‐trivial mathematical modelling challenges. Although there are literature reports of mathematical models to evaluate biofilm tolerance to biocides, none of these models have investigated biocide treatment of biofilms growing in interconnected porous media with flow. In this paper, we present a numerical investigation using a pore network model of biofilm growth, formation damage and biocide treatment. The model includes three phases (aqueous, adsorbed biofilm, and solid matrix), a single growth‐limiting nutrient and a single biocide dissolved in the water. Biofilm is assumed to contain a single species of microbe, in which each cell can be a viable persister, a viable non‐persister, or non‐viable (dead). Persisters describe small subpopulation of cells which are tolerant to biocide treatment. Biofilm tolerance to biocide treatment is regulated by persister cells and includes ‘innate’ and ‘biocide‐induced’ factors. Simulations demonstrate that biofilm tolerance to biocides can increase with biofilm maturity, and that biocide treatment alone does not reverse biofilm‐induced formation damage. Also, a successful application of biological permeability conformance treatment involving geologic layers with flow communication is more complicated than simply engineering the attachment of biofilm‐forming cells at desired sites.     

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.     

Anisotropic nutrient transport in three-dimensional single species bacterial biofilms

The ability for a biofilm to grow and function is critically dependent on the nutrient availability, and this in turn is dependent on the structure of the biofilm. This relationship is therefore an important factor influencing biofilm maturation. Nutrient transport in bacterial biofilms is complex; however, mathematical models that describe the transport of particles within biofilms have made three simplifying assumptions: the effective diffusion coefficient (EDC) is constant, the EDC is that of water, and/or the EDC is isotropic. Using a Monte Carlo simulation, we determined the EDC, both parallel to and perpendicular to the substratum, within 131 real, single species, three-dimensional biofilms that were constructed from confocal laser scanning microscopy images. Our study showed that diffusion within bacterial biofilms was anisotropic and depth dependent. The heterogeneous distribution of bacteria varied between and within species, reducing the rate of diffusion of particles via steric hindrance. In biofilms with low porosity, the EDCs for nutrient transport perpendicular to the substratum were significantly lower than the EDCs for nutrient transport parallel to the substratum. Here, we propose a reaction-diffusion model to describe the nutrient concentration within a bacterial biofilm that accounts for the depth dependence of the EDC.     

Simulation of growth and detachment in biofilm systems under defined hydrodynamic conditions

Detachment from biofilms was evaluated using a mixed culture biofilm grown on primary wastewater in a tube reactor. The growth of biofilms and the detachment of biomass from biofilms are strongly influenced by hydrodynamic conditions. In a long-term study, three biofilms were cultivated in a biofilm tube reactor. The conducted experiments of biofilm growth and detachment can be divided into three phases: 1) an exponential phase with a rapid increase of the biofilm thickness, 2) a quasi-steady-state with spontaneous fluctuation of the biofilm thickness between 500 and 1,200 microm in the investigated biofilm systems, and 3) a washout experiment with increased shear stress in three to four steps after several weeks of quasi-steady-state. Whereas the biofilm thickness during the homogeneous growth phase can be regarded constant throughout the reactor, it was found to be very heterogeneous during the quasi-steady-state and the washout experiments. Growth and detachment during all three phases was simulated with the same one-dimensional biofilm model. For each of the three phases, a different detachment rate model was used. During the homogeneous growth phase, detachment was modeled proportional to the biofilm growth rate. During the quasi-steady-state phase, detachment was described by random detachment events assuming a base biofilm thickness. Finally, the washout experiment was simulated with detachment being a function of the biofilm thickness before the increase of the shear stress.     

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.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

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).     

Quantification of local water and biomass in wild type PA01 biofilms by Confocal Raman Microspectroscopy

Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H stretching vibration band at 3450 cm(-1), (water), to that of the C-H stretching bands at 2950 cm(-1) (biomass), was used to estimate the relative biofilm water content. The quantification of biofilm water and biomass was based on calibration curves generated from protein solutions. Water/biomass ratios (W:BR) equivalent to that of a 30% (w/v) protein solution were observed within some biofilm colonies.     

A spider web strategy of type IV pili‐mediated migration to build a fibre‐like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms

Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)‐mediated bacterial migration leads to the formation of a fibre‐like Psl matrix. Deletion of T4P in wild type and flagella‐deficient strains results in loss of the Psl‐fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air‐liquid interface. Bacteria lacking T4P‐driven twitching motility including those that still express surface T4P are unable to form the Psl‐fibres. Formation of a Psl‐fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl‐fibres are likely formed by Psl released from bacteria during T4P‐mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P‐driven twitching motility. Furthermore, a radial‐pattern Psl‐fibre matrix is present in the middle of biofilms, a nutrient‐deprived region. These imply a plausible model for how bacteria respond to nutrient‐limited local environment to build a polysaccharide‐fibre matrix by T4P‐dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.     

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).