Sensitivity analysis of a biofilm model describing a one-stage completely autotrophic nitrogen removal (CANON) process.

A mathematical model for nitrification and anaerobic ammonium oxidation (ANAMMOX) processes in a single biofilm reactor (CANON) was developed. This model describes completely autotrophic conversion of ammonium to dinitrogen gas. Aerobic ammonium and nitrite oxidation were modeled together with ANAMMOX. The sensitivity of kinetic constants and biofilm and process parameters to the process performance was evaluated, and the total effluent concentrations were, in general, found to be insensitive to affinity constants. Increasing the amount of biomass by either increasing biofilm thickness and density or decreasing porosity had no significant influence on the total effluent concentrations, provided that a minimum total biomass was present in the reactor. The ANAMMOX process always occurred in the depth of the biofilm provided that the oxygen concentration was limiting. The optimal dissolved oxygen concentration level at which the maximum nitrogen removal occurred is related to a certain ammonium surface load on the biofilm. An ammonium surface load of 2 g N/m2. d, associated with a dissolved oxygen concentration level of 1.3 g O2/m3 in the bulk liquid and with a minimum biofilm depth of 1 mm seems a proper design condition for the one-stage ammonium removal process. Under this condition, the ammonium removal efficiency is 94% (82% for the total nitrogen removal efficiency) (30 degrees C). Better ammonium removal could be achieved with an increase in the dissolved oxygen concentration level, but this would strongly limit the ANAMMOX process and decrease total nitrogen removal. It can be concluded that a one-stage process is probably not optimal if a good nitrogen effluent is required. A two-stage process like the combined SHARON and ANAMMOX process would be advised for complete nitrogen removal.     

Continuum heterogeneous biofilm model--a simple and accurate method for effectiveness factor determination

We present a novel analytical approach to describe biofilm processes considering continuum variation of both biofilm density and substrate effective diffusivity. A simple perturbation and matching technique was used to quantify biofilm activity using the steady-state diffusion-reaction equation with continuum variable substrate effective diffusivity and biofilm density, along the coordinate normal to the biofilm surface. The procedure allows prediction of an effectiveness factor, η, defined as the ratio between the observed rate of substrate utilization (reaction rate with diffusion resistance) and the rate of substrate utilization without diffusion limitation. Main assumptions are that (i) the biofilm is a continuum, (ii) substrate is transferred by diffusion only and is consumed only by microorganisms at a rate according to Monod kinetics, (iii) biofilm density and substrate effective diffusivity change in the x direction, (iv) the substrate concentration above the biofilm surface is known, and (v) the substratum is impermeable. With this approach one can evaluate, in a fast and efficient way, the effect of different parameters that characterize a heterogeneous biofilm and the kinetics of the rate of substrate consumption on the behavior of the biological system. Based on a comparison of η profiles the activity of a homogeneous biofilm could be as much as 47.8% higher than that of a heterogeneous biofilm, under the given conditions. A comparison of η values estimated for first order kinetics and η values obtained by numerical techniques showed a maximum deviation of 1.75% in a narrow range of modified Thiele modulus values. When external mass transfer resistance, is also considered, a global effectiveness factor, η(0) , can be calculated. The main advantage of the approach lies in the analytical expression for the calculation of the intrinsic effectiveness factor η and its implementation in a computer program. For the test cases studied convergence was achieved quickly after four or five iterations. Therefore, the simulation and scale-up of heterogeneous biofilm reactors can be easily carried out.     

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.     

Predicting biofilm deformation with a viscoelastic phase-field model: Modeling and experimental studies

Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase-field (PF) continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, used as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The comparison between the experimental and modeling geometries, for selected horizontal cross sections, after fluid-driven deformation was good. The relative errors ranged from 3.2 to 21.1% for the synthetic biofilm and from 9.1 to 11.1% for the P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic PF biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

A porcine model of skin wound infected with a polybacterial biofilm

A clinically relevant porcine model of a biofilm-infected wound was established in 10 minipigs. The wounds of six experimental animals were infected with a modified polymicrobial Lubbock chronic wound biofilm consisting of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Bacillus subtilis. Four animals served as uninfected controls. The wounds were monitored until they had healed for 24 days. The biofilm persisted in the wounds up to day 14 and significantly affected healing. The control to infected healed wound area ratios were: 45%/21%, 66%/37%, and 90%/57% on days 7, 10 and 14, respectively. The implanted biofilm prolonged inflammation, increased necrosis, delayed granulation and impaired development of the extracellular matrix as seen in histological and gene expression analyses. This model provides a therapeutic one-week window for testing of anti-biofilm treatments and for research on the pathogenesis of wound infections in pig that is clinically the most relevant animal wound healing model.     

In vitro biofilm model for studying tongue flora and malodour

AIMS: To develop a perfusion biofilm system to model tongue biofilm microflora and their physiological response to sulfur-containing substrates (S-substrates) in terms of volatile sulfide compound (VSC) production. METHODS AND RESULTS: Tongue-scrape inocula were used to establish in vitro perfusion biofilms which were examined in terms of ecological composition using culture-dependent and independent (PCR-DGGE) approaches. VSC-specific activity of cells was measured by a cell suspension assay, using a portable industrial sulfide monitor which was also used to monitor VSC production from biofilms in situ. Quasi steady states were achieved by 48 h and continued to 96 h. The mean (+/-SEM) growth rate for 72-h biofilms (n=4) was micro=0.014 h(-1) (+/-0.005 h(-1)). Comparison of biofilms, perfusate and original inoculum showed their ecological composition to be similar (Pearson coefficient>0.64). Perfusate and biofilm cells derived from the same condition (co-sampled) were equivalent with regard to VSC-specific activities which were up-regulated in the presence of S-substrates. CONCLUSIONS: The model maintained a stable tongue microcosm suitable for studying VSC production; biofilm growth in the presence of S-substrates up-regulated VSC activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is apt for studying ecological and physiological aspects of oral biofilms and could be useful for screening inhibitory agents.     

Approximated solution of model for three-phase fluidized bed biofilm reactor in wastewater treatment

An approximated analytical solution of mathematical model for the three phase fluidized bed bioreactor (TFBBR) was proposed using the linearization technique to describe oxygen utilization rate in wastewater treatment. The validation of the model was done in comparison with the experimental results. Satisfactory agreement was obtained in the comparison of approximated analytical solution and numerical solution in the oxygen concentration profile of a TFBBR. The approximated solutions for three modes of the liquid phase flow were compared. The proposed model was able to predict the biomass concentration, dissolved oxygen concentration the height of efficient column, and the removal efficiency.     

Small and rough colony pseudomonas aeruginosa with elevated biofilm formation ability isolated in hospitalized patients

Pseudomonas aeruginosa is a key pathogen of nosocomial infection, and causes persistent infection in patients with specific diseases like cystic fibrosis (CF). It has been reported that patients affected with CF discharge, at a high frequency, small colony variants with high adherence ability. In routine laboratory testing, we found atypical small and rough type (SR) colony variants of P. aeruginosa. The SRs and the counterpart wild type (WT) colonies showed similar biochemical features, antimicrobial susceptibilities, pulsed-field gel electrophoresis (PFGE) profiles, serotypes, and twitching motilities. The biofilm formation abilities of all the SR colonies, however, were extremely elevated as compared to those of the counterpart WT colonies. The frequency of SR-positive patients was 3.1% of the P. aeruginosa-positive inpatients (5/160), and that of the SR isolates was 0.6% of the P. aeruginosa strains (6/970) isolated in our laboratory over a period of 6 months. The SR-positive patients did not have any common disease or particular antibiotics treatment. The PFGE profiles showed that the SRs and the counterpart WTs were identical to each other, and also that three of the five SR/WT pairs were clonally similar. The three pairs were recovered from the feces, urine, and endotracheal secretion, respectively, of three patients hospitalized in two distinct wards. The results suggest that P. aeruginosa spontaneously produced highly adherent SR colonies in hospitalized patients, and these colonies may tend to spread in a hospital.     

新生隐球菌生物膜动物感染模型的构建及其活性的检测

目的 构建一种简便易行的新生隐球菌生物膜感染动物模型.方法 采用大鼠皮下置管法构建新生隐球菌生物膜感染动物模型,并使用电子扫描显微镜观察新生隐球菌体内形成生物膜的结构;采用MTT法对获得的体内生物膜结构进行活性的检测.结果 成功构建了新生隐球菌生物膜大鼠感染模型;MTT法检测体内生物膜活性表明,随着体内培养时间的延长,生物膜活性增强,与直接镜检和电镜观察结果一致.结论 新生隐球菌生物膜大鼠模型简单易行,便于操作,对于研究新生隐球菌生物膜体内活性的研究具有一定的实际应用价值.     

Computational study of the drag and oscillatory movement of biofilm streamers in fast flows

Hydrodynamic conditions have a significant impact on the biofilm lifecycle. Not well understood is the fact that biofilms, in return, also affect the flow pattern. A decade ago, it was already shown experimentally that under fast flows, biofilm streamers form and oscillate with large amplitudes. This work is a first attempt to answer the questions on the mechanisms behind the oscillatory movement of the streamers, and whether this movement together with the special streamlined form of the streamers, have both a physical and biological benefit for biofilms. In this study, a state of the art two‐dimensional fluid–structure interaction model of biofilm streamers is developed, which implements a transient coupling between the fluid and biofilm mechanics. Hereby, it is clearly shown that formation of a Kármán vortex street behind the streamer body is the main source of the periodic oscillation of the streamers. Additionally it is shown that the formation of streamers reduces the fluid forces which biofilm surface experiences. Biotechnol. Bioeng. 2010; 105: 600–610. © 2009 Wiley Periodicals, Inc.     

Development and characterisation of a novel three-dimensional inter-kingdom wound biofilm model

Chronic diabetic foot ulcers are frequently colonised and infected by polymicrobial biofilms that ultimately prevent healing. This study aimed to create a novel in vitro inter-kingdom wound biofilm model on complex hydrogel-based cellulose substrata to test commonly used topical wound treatments. Inter-kingdom triadic biofilms composed of Candida albicans, Pseudomonas aeruginosa, and Staphylococcus aureus were shown to be quantitatively greater in this model compared to a simple substratum when assessed by conventional culture, metabolic dye and live dead qPCR. These biofilms were both structurally complex and compositionally dynamic in response to topical therapy, so when treated with either chlorhexidine or povidone iodine, principal component analysis revealed that the 3-D cellulose model was minimally impacted compared to the simple substratum model. This study highlights the importance of biofilm substratum and inclusion of relevant polymicrobial and inter-kingdom components, as these impact penetration and efficacy of topical antiseptics.     

Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.     

Combined discrete particle and continuum model predicting solid-state fermentation in a drum fermentor

The development of mathematical models facilitates industrial (large-scale) application of solid-state fermentation (SSF). In this study, a two-phase model of a drum fermentor is developed that consists of a discrete particle model (solid phase) and a continuum model (gas phase). The continuum model describes the distribution of air in the bed injected via an aeration pipe. The discrete particle model describes the solid phase. In previous work, mixing during SSF was predicted with the discrete particle model, although mixing simulations were not carried out in the current work. Heat and mass transfer between the two phases and biomass growth were implemented in the two-phase model. Validation experiments were conducted in a 28-dm3 drum fermentor. In this fermentor, sufficient aeration was provided to control the temperatures near the optimum value for growth during the first 45-50 hours. Several simulations were also conducted for different fermentor scales. Forced aeration via a single pipe in the drum fermentors did not provide homogeneous cooling in the substrate bed. Due to large temperature gradients, biomass yield decreased severely with increasing size of the fermentor. Improvement of air distribution would be required to avoid the need for frequent mixing events, during which growth is hampered. From these results, it was concluded that the two-phase model developed is a powerful tool to investigate design and scale-up of aerated (mixed) SSF fermentors.     

Modeling of an aerobic biofilm reactor with double-limiting substrate kinetics: bifurcational and dynamical analysis

A mathematical model of an aerobic biofilm reactor is presented to investigate the bifurcational patterns and the dynamical behavior of the reactor as a function of different key operating parameters. Suspended cells and biofilm are assumed to grow according to double limiting kinetics with phenol inhibition (carbon source) and oxygen limitation. The model presented by Russo et al. is extended to embody key features of the phenomenology of the granular‐supported biofilm: biofilm growth and detachment, gas–liquid oxygen transport, phenol, and oxygen uptake by both suspended and immobilized cells, and substrate diffusion into the biofilm. Steady‐state conditions and stability, and local dynamic behavior have been characterized. The multiplicity of steady states and their stability depend on key operating parameter values (dilution rate, gas–liquid mass transfer coefficient, biofilm detachment rate, and inlet substrate concentration). Small changes in the operating conditions may be coupled with a drastic change of the steady‐state scenario with transcritical and saddle‐node bifurcations. The relevance of concentration profiles establishing within the biofilm is also addressed. When the oxygen level in the liquid phase is <10% of the saturation level, the biofilm undergoes oxygen starvation and the active biofilm fraction becomes independent of the dilution rate. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Development and application of a polymicrobial, in vitro, wound biofilm model

Aims: The goal of this investigation was to develop an in vitro, polymicrobial, wound biofilm capable of supporting the growth of bacteria with variable oxygen requirements. Methods and Results: The strict anaerobe Clostridium perfringens was isolated by cultivating wound homogenates using the drip‐flow reactor (DFR), and a three‐species biofilm model was established using methicillin‐resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Cl. perfringens in the colony‐drip‐flow reactor model. Plate counts revealed that MRSA, Ps. aeruginosa and Cl. perfringens grew to 7·39 ± 0·45, 10·22 ± 0·22 and 7·13 ± 0·77 log CFU per membrane, respectively. The three‐species model was employed to evaluate the efficacy of two antimicrobial dressings, Curity? AMD and Acticoat?, compared to sterile gauze controls. Microbial growth on Curity? AMD and gauze was not significantly different, for any species, whereas Acticoat? was found to significantly reduce growth for all three species. Conclusions: Using the colony‐DFR, a three‐species biofilm was successfully grown, and the biofilms displayed a unique structure consisting of distinct layers that appeared to be inhabited exclusively or predominantly by a single species. Significance and Impact of the Study: The primary accomplishment of this study was the isolation and growth of an obligate anaerobe in an in vitro model without establishing an artificially anaerobic environment.     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.