Degradation of toluene by a mixed population of archetypal aerobes, microaerophiles, and denitrifiers: laboratory sand column experiment and multispecies biofilm model formulation

An experiment was conducted in a saturated sand column with three bacterial strains that have different growth characteristics on toluene, Pseudomonas putida F1 which degrades toluene only under aerobic conditions, Thauera aromatica T1 which degrades toluene only under denitrifying conditions, and Ralstonia pickettii PKO1 has a facultative nature and can perform nitrate-enhanced biodegradation of toluene under hypoxic conditions (DO <2 mg/L). Steady-state concentration profiles showed that oxygen and nitrate appeared to be utilized simultaneously, regardless of the dissolved oxygen concentration and the results from fluorescent in-situ hybridization (FISH) indicated that PKO1 maintained stable cells numbers throughout the column, even when the pore water oxygen concentration was high. Since PKO1's growth rate under aerobic condition is much lower than that of F1, except under hypoxic conditions, these observations were not anticipated. Therefore these observations require a mechanistic explanation that can account for localized low oxygen concentrations under aerobic conditions. To simulate the observed dynamics, a multispecies biofilm model was implemented. This model formulation assumes the formation of a thin biofilm that is composed of the three bacterial strains. The individual strains grow in response to the substrate and electron acceptor flux from bulk fluid into the biofilm. The model was implemented such that internal changes in bacterial composition and substrate concentration can be simulated over time and space. The model simulations from oxic to denitrifying conditions compared well to the experimental profiles of the chemical species and the bacterial strains, indicating the importance of accounting for the biological activity of individual strains in biofilms that span different redox conditions.     

Modeling and analysis of layered stationary anaerobic granular biofilms

A model that portrays substrate profiles in a steady-state multispecies granular biofilm is developed and coupled with a biofilm detachment model. The model accounts for glucose, propionate, hydrogen, and acetate transformations performed by three bacterial trophic groups: acidogens, syntrophic bacterial consortia, and methanogens. This model adequately describes the phenomenon of propionate degradation under thermodynamically unfavorable bulk hydrogen concentrations. Also suggested is the superiority of the layered biofilm structure over homogeneous distribution of the trophic groups for anaerobic degradation of organic compounds. Furthermore, model analysis suggests that with increasing bulk glucose concentration biofilm thickness reaches a maximum that is then followed by biofilm disintegration. These results may have an important impact on the design and control of upflow anaerobic sludge bed reactors. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 122-130, 1997.     

Modeling biocide action against biofilms

A phenomenological model of biocide action against microbial biofilms was derived. Processes incorporated in the model include bulk flow in and out of a well-mixed reactor, transport of dissolved species into the biofilm, substrate consumption by bacterial metabolism, bacterial growth, advection of cell mass within the biofilm, cell detachment from the biofilm, cell death, and biocide concentration-dependent disinfection. Simulations were performed to analyze the general behavior of the model and to perform preliminary sensitivity analysis to identify key input parameters. The model captured several general features of antimicrobial agent action against biofilms that have been observed widely by experimenters and practitioners. These included (1) rapid disinfection followed by biofilm regrowth, (2) slower detachment than disinfection, and (3) reduced susceptibility of microorganisms in biofilms. The results support the plausibility of a mechanism of biofilm resistance in which the biocide is neutralized by reaction with biofilm constituents, leading to a reduction in the bulk biocide concentration and, more significantly, biocide concentration gradients within the biofilm. Sensitivity experiments and analyses identified which input parameters influence key response variables. Each of three response variables was sensitive to each of the five input parameters, but they were most sensitive to the initial biofilm thickness and next most sensitive to the biocide disinfection rate coefficient. Statistical regression modeling produced simple equations for approximating the response variables for situations within the range of conditions covered by the sensitivity experiment. The model should be useful as a tool for studying alternative biocide control strategies. For example, the simulations suggested that a good interval between pulses of biocide is the time to minimum thickness. (c) 1996 John Wiley & Sons, Inc.     

Construction and analysis of fractional multifactorial designs to study attachment strength and transfer of Listeria monocytogenes from pure or mixed biofilms after contact with a solid model food

The aim of this study was to establish which of seven factors influence the adhesion strength and hence bacterial transfer between biofilms containing Listeria monocytogenes (pure and two-species biofilms) and tryptone soya agar (TSA) as a solid organic surface. The two-species biofilms were made of L. monocytogenes and one of the following species of bacteria: the nonpathogenic organisms Kocuria varians, Pseudomonas fluorescens, and Staphylococcus sciuri and CCL 63, an unidentified gram-negative bacterium isolated from the processing plant environment. We used biofilms prepared under conditions simulating open surfaces in meat-processing sites. The biofilm's adhesion strength and population were evaluated by making 12 contacts on a given whole biofilm (4.5 cm(2)), using a new slice of a sterilized TSA cylinder for each contact, and plotting the logarithm CFU . cm(-2) detached by each contact against the contact number. Three types of detachment kinetics were observed: biphasic kinetics, where the first slope may be either positive or negative, and monophasic kinetics. The bacteria that resisted a chlorinated alkaline product and a glutaraldehyde- and quaternary ammonium-based disinfectant had greater adhesion strengths than those determined for untreated biofilms. One of the four non-Listeria strains studied, Kocuria varians CCL 56, favored both the attachment and detachment of L. monocytogenes. The stainless steel had smaller bacterial populations than polymer materials, and non-Listeria bacteria adhered to it less strongly. Our results helped to evaluate measures aimed at controlling the immediate risk, linked to the presence of a large number of CFU in a foodstuff, and the delayed risk, linked to the persistence of L. monocytogenes and the occurrence of slightly contaminated foods that may become dangerous if L. monocytogenes multiplies during storage. Cleaning and disinfection reduce the immediate risk, while reducing the delayed risk should be achieved by lowering the adhesion strength, which the sanitizers used here cannot do at low concentrations.     

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).     

A modeling study of anaerobic biofilm systems: I. Detailed biofilm modeling

A detailed model acetate-utilizing methanogenic biofilms accounting for the diffusion of neutral and ionic species, chemical equilibrium, electroneutrality, gas production within the biofilm, pH-dependent Monod kinetics, and the presence of a concentration boundary layer is presented. The model qualitatively fits the pH profiles that are reported for acetate-utilizing methanogenic aggregates. A sensitivity analysis on the biological parameters showed that the flux of acetate is sensitive to the maximum utilization rate, half-saturation constant, and biofilm density for the bulk conditions investigated. Criteria when traditional biofilm models can be used to predict the flux of acetate into the biofilm are established. If the maximum pH change predicted using a hypothetical system is within +/-0.05, the traditional model predicts the flux to within +/-5% of the value calculated with the model developed in this study. (c) 1995 John Wiley & Sons, Inc.     

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.     

A semi-quantitative approach to assess biofilm formation using wrinkled colony development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis, and Gram-negative bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Vibrio fischeri. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect, while strains exhibiting increased biofilm phenotypes are enhanced for colonization. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.     

Is there a role for quorum sensing signals in bacterial biofilms?

Bacteria form multicellular biofilm communities on most surfaces. Genetic analysis of biofilm formation has led to the proposal that extracellular signals and quorum-sensing regulatory systems are essential for differentiated biofilms. Although such a model fits the concept of density-driven cell-cell communication and appear to describe biofilm development in several bacterial species and conditions, biofilm formation is multifactorial and complex. Hydrodynamics, nutrient load and intracellular carbon flux have major impacts, presumably by altering the expression of cellular traits essential for bacterial adaptation during the different stages of biofilm formation. Hence, differentiated biofilms may also be the net result of many independent interactions, rather than being determined by a particular global quorum sensing system.     

Immigration and emigration of Burkholderia cepacia and Pseudomonas aeruginosa between and within mixed biofilm communities

AIMS: To investigate the dynamics of binary culture biofilm formation through use of both the Sorbarod model of biofilm growth and the constant depth film fermenter (CDFF). METHODS AND RESULTS: Pseudo steady-state biofilm cultures of laboratory and clinical strains of Pseudomonas aeruginosa, selected on the basis of their ability to produce a Burkholderia cepacia growth-inhibitory substance, were established on Sorbarod filters and challenged with corresponding planktonic grown cultures of B. cepacia. Reverse challenges were also conducted. Both B. cepacia and P. aeruginosa were able to form steady-state monoculture biofilms after 48 h growth. When steady-state biofilms of B. cepacia NTCT 10661 were challenged with planktonically grown P. aeruginosa PAO1 known to produce a B. cepacia growth-inhibitory substance, the immigrant population was rapidly and almost completely bound to the biofilm, displacing B. cepacia. By contrast, established biofilms of P. aeruginosa PAO1 resisted immigration of B. cepacia 10661. Similar experiments conducted with a nongrowth inhibitory substance producing clinical pairing of P. aeruginosa 313113 and B. cepacia 313113 led to the formation of stable, mixed biofilm populations in both instances. Moreover, co-inoculation with these clinical isolates resulted in a stable, mixed steady-state biofilm. Similar observations were made for biofilms generated in CDFFs. In such instances following pan-swapping between two monoculture CDFFs, B. cepacia 313113 was able to integrate into an established P. aeruginosa 313113 biofilm to form a stable binary biofilm. CONCLUSIONS: Establishment of a mixed species community follows a specific sequence of inoculation that may either be due to some degree of match between co-colonizers or that P. aeruginosa predisposes uncolonized sections of the surface to permit B. cepacia colonization. SIGNIFICANCE AND IMPACT OF THE STUDY: Colonization of a surface with one bacterial species confers colonization resistance towards other species. Disinfection of a surface might well increase the probability of pathogen harbourage.     

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).     

Three-Dimensional Numerical Simulations of Biofilm Dynamics with Quorum Sensing in a Flow Cell

We develop a multiphasic hydrodynamic theory for biofilms taking into account interactions among various bacterial phenotypes, extracellular polymeric substance (EPS), quorum sensing (QS) molecules, solvent, and antibiotics. In the model, bacteria are classified into down-regulated QS, up-regulated QS, and non-QS cells based on their QS ability. The model is first benchmarked against an experiment yielding an excellent fit to experimental measurements on the concentration of QS molecules and the cell density during biofilm development. It is then applied to study development of heterogeneous structures in biofilms due to interactions of QS regulation, hydrodynamics, and antimicrobial treatment. Our 3D numerical simulations have confirmed that (i). QS is beneficial for biofilm development in a long run by building a robust EPS population to protect the biofilm; (ii). biofilms located upstream can induce QS downstream when the colonies are close enough spatially; (iii). QS induction may not be fully operational and can even be compromised in strong laminar flows; (v). the hydrodynamic stress alters the biofilm morphology. Through further numerical investigations, our model suggests that (i). QS-regulated EPS production contributes to the structural formation of heterogeneous biofilms; (ii) QS down-regulated cells tend to grow at the surface of the biofilm while QS up-regulated ones tend to grow in the bulk; (iii) when nutrient supply is sufficient, QS induction might be more effective upstream than downstream; (iv) QS may be of little benefit in a short timescale in term of fighting against invading strain/species; (v) the material properties of biomass (bacteria and EPS) have strong impact on the dilution of QS molecules under strong shear flow. In addition, with this modeling framework, hydrodynamic details and rheological quantities associated with biofilm formation under QS regulation can be resolved.     

A general description of detachment for multidimensional modelling of biofilms

A general method for describing biomass detachment in multidimensional biofilm modelling is introduced. Biomass losses from processes acting on the entire surface of the biofilm, such as erosion, are modelled using a continuous detachment speed function F(det). Discrete detachment events, i.e. sloughing, are implicitly derived from simulations. The method is flexible to allow F(det) to take several forms, including expressions dependent on any state variables such as the local biofilm density. This methodology for biomass detachment was integrated with multidimensional (2D and 3D) particle-based multispecies biofilm models by using a novel application of the level set method. Application of the method is illustrated by trends in the dynamics of biofilms structure and activity derived from simulations performed on a simple model considering uniform biomass (case study I) and a model discriminating biomass composition in heterotrophic active mass, extracellular polymeric substances (EPS) and inert mass (case study II). Results from case study I demonstrate the effect of applied detachment forces as a fundamental factor influencing steady-state biofilm activity and structure. Trends from experimental observations reported in literature were correctly described. For example, simulation results indicated that biomass sloughing is reduced when erosion forces are increased. Case study II illustrates the application of the detachment methodology to systems with non-uniform biomass composition. Simulations carried out at different bulk concentrations of substrate show changes in biofilm structure (in terms of shape, density and spatial distribution of biomass components) and activity (in terms of oxygen and substrate consumption) as a consequence of either oxygen-limited or substrate-limited growth.     

Preliminary characterization of thin biofilms by optical microscopy

A simple non‐invasive technique has been used that employs conventional optical microscopy and a glass flow cell to observe biofilms formed on opaque thin substrata. The technique allows the roughness of the biofilm and the substratum to be evaluated, and the biofilm thickness to be easily measured. The biofilm density may be quantified through colour gradients. In addition, some details of biofilm growth processes like the formation of water channels and pores, and interactions between planktonic and sessile cells can be visualized. Results related to the development of thin biofilms and their response to the environment under different conditions are reported. Pure and mixed microbial cultures and different solid substrata were assessed.     

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Development and experimental evaluation of a steady-state, multispecies biofilm model

A steady-state model for quantifying the space competition in multispecies biofilms is developed. The model includes multiple active species, inert biomass, substrate utilization and diffusion within the biofilm, external mass transport, and detachment phenomena. It predicts the steady-state values of biofilm thickness, species distribution, and substrate fluxes. An experimental evaluation is carried out in completely mixed biofilm reactors in which slow-growing nitrifying bacteria compete with acetate-utilizing heterotrophs. The experimental results show that the model successfully describes the space competition. In particular, increasing acetate concentrations causes NH(4) (+)-N fluxes to decrease, because nitrifiers are forced deeper into the biofilm, where they experience greater mass-transport resistance.     

Effect of Periodic Disinfection on Persisters in a One-Dimensional Biofilm Model

It is well known that disinfection methods that successfully kill suspended bacterial populations often fail to eliminate bacterial biofilms. Recent efforts to understand biofilm survival have focused on the existence of small, but very tolerant, subsets of the bacterial population termed persisters. In this investigation, we analyze a mathematical model of disinfection that consists of a susceptible-persister population system embedded within a growing domain. This system is coupled to a reaction-diffusion system governing the antibiotic and nutrient. We analyze the effect of periodic and continuous dosing protocols on persisters in a one-dimensional biofilm model, using both analytic and numerical method. We provide sufficient conditions for the existence of steady-state solutions and show that these solutions may not be unique. Our results also indicate that the dosing ratio (the ratio of dosing time to period) plays an important role. For long periods, large dosing ratios are more effective than similar ratios for short periods. We also compare periodic to continuous dosing and find that the results also depend on the method of distributing the antibiotic within the dosing cycle.     

Effect of different concentrations of ortho-phthalaldehyde on biofilms formed by Pseudomonas fluorescens under different flow conditions

The effectiveness of different concentrations of ortho-phthalaldehyde (OPA) in controlling biofilms of Pseudomonas fluorescens formed on stainless steel slides, using flow cell reactors under laminar and turbulent flow, was investigated by determining the variation in mass and respiratory activity. The physical stability of the biofilm with and without exposure to OPA was studied in a rotating device as variation in the mass of the biofilm on the surface after exposure to different rotation velocities. The activity of OPA against bacterial suspended cultures was evaluated in the presence and absence of bovine serum albumin (BSA) in order to evaluate the interference of proteins on the activity of the biocide. The results showed that biofilms formed under different flow conditions had different properties and reacted differently after biocide application. Biofilms formed under laminar flow were more easily inactivated than those formed under turbulent conditions. However, OPA did not promote the detachment of biofilms from the surface. The exposure of biofilms to different shear stress conditions after OPA treatment enhanced removal from the surface, indicating that OPA may weaken the biofilm matrix. The biocide was more effective on suspended cells than on cells grown in biofilms. This fact may be explained by the reaction of the biocide with proteins of the polymeric matrix of the biofilm as suggested by the significant reduction of biocide action on suspended cells in the presence of BSA.     

Influence of abrasion on biofilm detachment: evidence for stratification of the biofilm

The objective of this paper was to understand the detachment of multispecies biofilm caused by abrasion. By submitting a biofilm to different abrasion strengths (collision of particles), stratification of biofilm cohesion could be highlighted and related to stratification of biofilm bacterial communities using the PCR-SSCP fingerprint method. The biofilm comprised a thick top layer, weakly cohesive and composed of one dominant species, and a thin basal layer, strongly cohesive and composed of a more diverse population. These observations suggest that microbial composition of biofilms may be an important parameter in understanding biofilm detachment.     

A Multiscale Agent-Based Model for the Investigation of <Emphasis Type="Italic">E. coli</Emphasis> K12 Metabolic Response During Biofilm Formation

Bacterial biofilm formation is an organized collective response to biochemical cues that enables bacterial colonies to persist and withstand environmental insults. We developed a multiscale agent-based model that characterizes the intracellular, extracellular, and cellular scale interactions that modulate Escherichia coli MG1655 biofilm formation. Each bacterium’s intracellular response and cellular state were represented as an outcome of interactions with the environment and neighboring bacteria. In the intracellular model, environment-driven gene expression and metabolism were captured using statistical regression and Michaelis–Menten kinetics, respectively. In the cellular model, growth, death, and type IV pili- and flagella-dependent movement were based on the bacteria’s intracellular state. We implemented the extracellular model as a three-dimensional diffusion model used to describe glucose, oxygen, and autoinducer 2 gradients within the biofilm and bulk fluid. We validated the model by comparing simulation results to empirical quantitative biofilm profiles, gene expression, and metabolic concentrations. Using the model, we characterized and compared the temporal metabolic and gene expression profiles of sessile versus planktonic bacterial populations during biofilm formation and investigated correlations between gene expression and biofilm-associated metabolites and cellular scale phenotypes. Based on our in silico studies, planktonic bacteria had higher metabolite concentrations in the glycolysis and citric acid cycle pathways, with higher gene expression levels in flagella and lipopolysaccharide-associated genes. Conversely, sessile bacteria had higher metabolite concentrations in the autoinducer 2 pathway, with type IV pili, autoinducer 2 export, and cellular respiration genes upregulated in comparison with planktonic bacteria. Having demonstrated results consistent with in vitro static culture biofilm systems, our model enables examination of molecular phenomena within biofilms that are experimentally inaccessible and provides a framework for future exploration of how hypothesized molecular mechanisms impact bulk community behavior.