Particle-based multidimensional multispecies biofilm model

In this paper we describe a spatially multidimensional (two-dimensional [2-D] and three-dimensional [3-D]) particle-based approach for modeling the dynamics of multispecies biofilms growing on multiple substrates. The model is based on diffusion-reaction mass balances for chemical species coupled with microbial growth and spreading of biomass represented by hard spherical particles. Effectively, this is a scaled-up version of a previously proposed individual-based biofilm model. Predictions of this new particle-based model were quantitatively compared with those obtained with an established one-dimensional (1-D) multispecies model for equivalent problems. A nitrifying biofilm containing aerobic ammonium and nitrite oxidizers, anaerobic ammonium oxidizers, and inert biomass was chosen as an example. The 2-D and 3-D models generally gave the same results. If only the average flux of nutrients needs to be known, 2-D and 1-D models are very similar. However, the behavior of intermediates, which are produced and consumed in different locations within the biofilm, is better described in 2-D and 3-D models because of the multidirectional concentration gradients. The predictions of 2-D or 3-D models are also different from those of 1-D models for slowly growing or minority species in the biofilm. This aspect is related to the mechanism of biomass spreading or advection implemented in the models and should receive more attention in future experimental studies.     

A new method to solve a non-steady-state multispecies biofilm model

A transient multispecies model for quantifying microbial space competition in biofilm is derived from existing models, introducing a new approach to biomass detachment modelling. This model includes inert biomass, substrate diffusion and utilization rate within the biofilm and diffusional layers. It predicts the evolution of biofilm thickness, bulk substrate concentration, species distribution and substrate concentration within the biofilm. A zero-dimensional transient model is described. Its steady-state solution is used to set up initial conditions of the one-dimensional model and case computation towards steady-state solution. Some numerical tools have been developed, enabling fast computation on microcomputers. Simulations show the validity of a zero-dimensional model and perturbated systems are also simulated. Simulations with experimental data give acceptable results.     

All together now: experimental multispecies biofilm model systems

Studies of microorganisms have traditionally focused on single species populations, which have greatly facilitated our understanding of the genetics and physiology that underpin microbial growth, adaptation and biofilm development. However, given that most microorganisms exist as multispecies consortia, the field is increasingly exploring microbial communities using a range of technologies traditionally limited to populations, including meta‐omics based approaches and high resolution imaging. The experimental communities currently being explored range from relatively low diversity, for example, two to four species, to significantly more complex systems, comprised of several hundred species. Results from both defined and undefined communities have revealed a number of emergent properties, including improved stress tolerance, increased biomass production, community level signalling and metabolic cooperation. Based on results published to date, we submit that community‐based studies are timely and increasingly reveal new properties associated with multispecies consortia that could not be predicted by studies of the individual component species. Here, we review a range of defined and undefined experimental systems used to study microbial community interactions.     

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.     

Development of a multispecies periodontal biofilm model within a stirred bioreactor

Abstract

The objective of this work was to develop a subgingival biofilm model using a stirred bioreactor. Discs of bovine teeth were adapted to a stirred bioreactor filled with a culture medium containing bacterial species associated with periodontal health or disease. After anaerobic incubation, the biofilms growing on the substratum surfaces were collected and analyzed. The mean number of Colony-forming Units (CFUs) varied, but with no difference between 3 and 7?days of biofilm formation (p?>?0.05). Scanning Electron Microscopy (SEM) analysis showed a uniform biofilm layer covering the cement layer of the root surface containing bacteria with diverse morphology. In checkerboard DNA-DNA hybridization, bacterial species were identified in both biofilms. In conclusion, a subgingival biofilm model was developed using a stirred bioreactor, allowing the in vitro reproduction of complex microbial communities. This is an advanced model that may be useful to mimic complex clinical periodontal biofilms.     

A multispecies dependent double‐observer model: A new method for estimating multispecies abundance

Conservation of biological communities requires accurate estimates of abundance for multiple species. Recent advances in estimating abundance of multiple species, such as Bayesian multispecies N‐mixture models, account for multiple sources of variation, including detection error. However, false‐positive errors (misidentification or double counts), which are prevalent in multispecies data sets, remain largely unaddressed. The dependent‐double observer (DDO) method is an emerging method that both accounts for detection error and is suggested to reduce the occurrence of false positives because it relies on two observers working collaboratively to identify individuals. To date, the DDO method has not been combined with advantages of multispecies N‐mixture models. Here, we derive an extension of a multispecies N‐mixture model using the DDO survey method to create a multispecies dependent double‐observer abundance model (MDAM). The MDAM uses a hierarchical framework to account for biological and observational processes in a statistically consistent framework while using the accurate observation data from the DDO survey method. We demonstrate that the MDAM accurately estimates abundance of multiple species with simulated and real multispecies data sets. Simulations showed that the model provides both precise and accurate abundance estimates, with average credible interval coverage across 100 repeated simulations of 94.5% for abundance estimates and 92.5% for detection estimates. In addition, 92.2% of abundance estimates had a mean absolute percent error between 0% and 20%, with a mean of 7.7%. We present the MDAM as an important step forward in expanding the applicability of the DDO method to a multispecies setting. Previous implementation of the DDO method suggests the MDAM can be applied to a broad array of biological communities. We suggest that researchers interested in assessing biological communities consider the MDAM as a tool for deriving accurate, multispecies abundance estimates.     

A model of biofilm detachment

A general mathematical framework for modeling biofilm detachment is presented. The approach is founded on a material balance on biomass that equates the detachment rate to the product of a detachment frequency and a detaching particle mass. The model provides a theoretical basis for deriving many of the empirical detachment rate expressions in common use and can thus lend some insight into their physical and biological significance. By allowing for variation in the detachment frequency with depth in the biofilm, the model permits derivation of detachment expressions that reflect a dependence on chemical or physiological gradients in the biofilm. Analysis of literature data sets from two different biofilm systems suggests, in both cases, that detachment is a growth-associated phenomenon. (c) 1993 John Wiley & Sons, Inc.     

The serine protease Esperase HPF inhibits the formation of multispecies biofilm

The antifouling (AF) potential of the serine protease Esperase HPF (subtilisin) was evaluated for the ability to prevent the formation of a four-species bacterial biofilm. The effects of enzyme activity, time and application of the enzyme were tested on the density and the oxidative metabolism of biofilm developed in microtiter wells. Esperase HPF did not inhibit the oxidative metabolism of the bacterial biofilm or planktonic growth, but the enzyme inhibited biofilm formation by its proteolytic activity as inactivated enzyme had no effect. The effective enzyme concentration was determined over a period of 72 h, as by then all the tested concentrations inhibited biofilm formation maximally. The effective concentrations of the enzymes in solution were the same regardless of time of application (ie before or after biofilm formation), but immobilisation of the enzymes caused a lower effective concentration. Esperase HPF is an attractive alternative to the biocidal compounds used in AF coatings today.     

Low‐abundant species facilitates specific spatial organization that promotes multispecies biofilm formation

Microorganisms frequently co‐exist in matrix‐embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four‐species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four‐species biofilm. Interestingly, the presence of the low‐abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four‐species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four‐species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four‐species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

The Freter model: A simple model of biofilm formation

A simple, conceptual model of biofilm formation, due to R. Freter et al. (1983), is studied analytically and numerically in both CSTR and PFR. Two steady state regimes are identified, namely, the complete washout of the microbes from the reactor and the successful colonization of both the wall and bulk fluid. One of these is stable for any particular set of parameter values and sharp and explicit conditions are given for the stability of each. The effects of adding an anti-microbial agent to the CSTR are examined.Supported by NSF Grant DMS 0107439 and UTA Grant REP 14748717Supported by NSF Grant DMS 0107160     

A continuous culture biofilm model of cariogenic responses

AIMS: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization. METHODS AND RESULTS: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced 'pH-cycling' conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to 'white-spot' lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy. CONCLUSION: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization. SIGNIFICANCE AND IMPACT OF THE STUDY: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.     

The growth and resistance to sodium hypochlorite of Listeria monocytogenes in a steady-state multispecies biofilm

A constant-depth film fermenter (CDFF) was used to culture a steady-state multispecies biofilm consisting of one strain each of Listeria monocytogenes, Pseudomonas fragi and Staphylococcus xylosus. These bacteria were initially grown together in a conventional chemostat to achieve a steady state before being inoculated into the CDFF over an 18-h period. A dilute tryptone soya broth (TSB) medium was supplied to the CDFF and the biofilm allowed to develop over a 28-d period. This mature biofilm was then subjected to increasing levels of sodium hypochlorite solution to measure any antimicrobial effect. The three organisms were seen to reach a steady state after 6 d in the chemostat before being transferred to the CDFF where the mature multispecies biofilm reached steady state at 17 d. Listeria monocytogenes in both planktonic and biofilm growth stabilized at 1. 8 and 1.5%, respectively, of the total plate counts, while Ps. fragi and Staph. xylosus were the predominant organisms in the biofilm at 59% and 39.5%, respectively, of the total microbial population. Steady-state biofilms in the CDFF were exposed to increasing strengths of sodium hypochlorite; 200, 500 and 1000 p.p.m. free chlorine, but a substantial two-log cycle drop in bacterial numbers was only achieved at 1000 p.p.m. free chlorine. In planktonic culture all three organisms were completely eliminated when exposed to 10 p.p.m. free chlorine for a 30-s period.     

A proposed transient model for cometabolism in biofilm systems

A dynamic model was developed to describe the behaviour of primary and secondary substrates in a biofilm reactor. The model incorporates structured kinetics to describe the generation and consumption of reducing power in the catabolic and respiratory subsystems, respectively. Secondary substrate transformation through oxygenolytic or reductive mechanisms can be modelled under either single or dual limitation of the electron donor and electron acceptor substrates. An example simulation of a theoretical biofilm system was performed.     

A biofilm model to understand the onset of sulfate reduction in denitrifying membrane biofilm reactors

This work presents a multispecies biofilm model that describes the co‐existence of nitrate‐ and sulfate‐reducing bacteria in the H₂‐based membrane biofilm reactor (MBfR). The new model adapts the framework of a biofilm model for simultaneous nitrate and perchlorate removal by considering the unique metabolic and physiological characteristics of autotrophic sulfate‐reducing bacteria that use H₂ as their electron donor. To evaluate the model, the simulated effluent H₂, UAP (substrate‐utilization‐associated products), and BAP (biomass‐associated products) concentrations are compared to experimental results, and the simulated biomass distributions are compared to real‐time quantitative polymerase chain reaction (qPCR) data in the experiments for parameter optimization. Model outputs and experimental results match for all major trends and explain when sulfate reduction does or does not occur in parallel with denitrification. The onset of sulfate reduction occurs only when the nitrate concentration at the fiber's outer surface is low enough so that the growth rate of the denitrifying bacteria is equal to that of the sulfate‐reducing bacteria. An example shows how to use the model to design an MBfR that achieves satisfactory nitrate reduction, but suppresses sulfate reduction. Biotechnol. Bioeng. 2013; 110: 763–772. © 2012 Wiley Periodicals, Inc.