Biofilm reactor calibration for in vitro investigation of composite biodegradation

Kostadin Georgiev; Ivan Filipov; Aleksandra Pecheva

doi:10.3897/folmed.64.e61540

Original Article

Biofilm reactor calibration for in vitro investigation of composite biodegradation

Kostadin Georgiev^‡, Ivan Filipov^‡, Aleksandra Pecheva^‡

‡ Medical University of Plovdiv, Plovdiv, Bulgaria

Corresponding author: Kostadin Georgiev ( kostadin.georgiev@mu-plovdiv.bg )

This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation: Georgiev K, Filipov I, Pecheva A (2022) Biofilm reactor calibration for in vitro investigation of composite biodegradation. Folia Medica 64(2): 248-251. https://doi.org/10.3897/folmed.64.e61540

Abstract

Introduction: The majority of biodegradation studies of composite materials use simplified models of microbial biofilm despite the apparent diversity of the oral microbiota. The use of in vitro systems of “artificial mouth” design is a step towards clarifying the synergistic effect that microbial plaque and human saliva have on composite degradation.

Aim: Establishment of functional parameters for in vitro reproduction of oral biofilms via biofilm reactor systems.

Materials and methods: The CDC Biofilm Reactor system consists of eight polypropylene sticks. The rod cover and the retaining plates are mounted in a 1-dm glass cylinder with an outlet side opening. The laboratory bioreactor has a working volume of 340 ml. The device is equipped with a four-blade magnetic stirrer. The system also includes gauging appliances and executive mechanisms for controlling and adjusting the basic parameters of the process.

Results: Determination of the operating volume of the reactor is performed prior to the experiment along with the time of reach and stabilization of the set temperature in the design which is 60 min at 120 rpm. A mathematical model is used to calculate the rate of delivery of growth medium - 11 millilitres per minute. The bioreactor is sterilized by 0.3% neomycin solution for 24 hours. Prior to the experiment the system is cleansed (via passage) with sterile water for 60 minutes.

Conclusions: The pre-calibration of a bioreactor system allows specification and refinement of its working parameters, thus engaging for accurate reproduction of the environmental conditions in the oral cavity.

Keywords

artificial mouth, bioreactor, in vitro, oral biofilm

Introduction

Oral environment is inhabited by more than 700 bacterial taxa. The hard and soft tissues in the mouth are a natural substrate used by the oral microbiota to form complex and heterogenic microcosm biofilms.^[1–3]

The recent focus of contemporary cariology research is creating controllable and highly reproductive biofilm culture models via open and closed test systems. The test models based on a closed system accommodate scarce bacterial diversity and a limited supply of nutrients despite being simple to perform and cost-effective. On the other hand, open system models are more complex involving diverse bacterial spectrum and simultaneously ensuring continuous supply of fresh medium, metabolites removal, and culture liquid. Shortcomings of this type of model design are its technique sensitivity, cost, and proficiency to perform. However, open system biofilm models provide better regulation of investigation parameters and thus are rendered superior to closed system designs.^[4]

The majority of studies in the literature implement simplified models of microbial biofilm despite the apparent diversity of oral microbiota. The use of artificial mouth-based in vitro design is a step towards a better understanding of the bacterial plaque-saliva complex and its combined effects on the biodegradation of composite materials.

AIM

The aim of this study was to establish the functional parameters for in vitro reproduction of oral biofilms via biofilm reactor systems.

Materials and methods

The CDC Biofilm Reactor system used in our experiment can accurately simulate an in vivo environment using computer-controlled facilities (Fig. 1). It consists of eight polypropylene sticks - biofilm plate holders hinged on a polyethylene lid. The rod cover and the retaining plates are mounted in a 1-dm glass cylinder with an outlet side opening (Fig. 2). The laboratory bioreactor has a working volume of 340 ml. The device is equipped with a four-blade magnetic stirrer. The system also includes gauging appliances and executive mechanisms for controlling and adjusting the basic parameters of the process. Acidity is monitored through a pH-electrode and a micro-reference electrode. Calibration processes are performed with distilled water. Environmental variables are easily controlled in the bioreactor. This allows analysis of the biofilm during its development without contaminating other samples.

Determination of the operating volume of the reactor is performed prior to the experiment along with the time of reach and stabilization of the set temperature (60 min at 120 rpm). A mathematical model is used to calculate the rate of delivery of growth medium (11 millilitres per minute). Prior to the experiment, the bioreactor is sterilized by 0.3% neomycin solution for 24 hours and cleansed (via passage) with sterile water for 60 minutes.

Figure 1.

The CDC Biofilm Reactor system simulates an in vivo environment using computer-controlled facilities.

Figure 2.

The rod cover and the retaining plates mounted in a 1-dm glass cylinder with an outlet side opening.

Results

Table 1 presents the results of this study.

Table 1.

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Results of the present study

Parameter	Value
Time of reach of set temperature (36.6°C)	60 min
Stirring rate	120 rpm
Flow of medium (BMM, species-dependent)	11 ml/min
Cultivation cycle	72 h (species-dependent)
Working volume	340 ml
Set acidity of the environment (pH)	7 pH

Discussion

Laboratory microbial culture models simulate the oral environment for cariology studies. Unlike in vivo studies, laboratory simulations do not face problems related to the uncontrollable fluctuations of the oral environment.^[5,6] Two complementary microbiological approaches can be taken to generate biofilm in microbial culture models. One of these approaches investigates the evolution of a plaque microcosm from natural oral microflora.^[7] The other approach is the construction of defined-species biofilm consortia with major plaque species, or a mixture of different species of the acquired oral bacteria, such as the American Type Culture Collection (ACTT) bacteria. The designs of laboratory microbial culture models vary according to the purpose of the laboratory studies – these models are classified as closed and open systems. Each system is a compromise between the reality of the in vivo ecosystem and the simplification of the system. However, a well-designed model and a study allow researchers to obtain precise and useful results.^[7]

The closed system

Microbial culture models in the closed system have a finite supply of nutrients. The growth rates of the biofilm are rapid at the beginning of the cultivation cycle when there is plethora of nutrients. However, this is rarely observed in the in vivo growth of biofilm.^[8,9] The growth conditions will change significantly following the consumption of nutrients and the accumulation of metabolic products. Hence, the physiological and biological properties of the biofilm are not comparable with those observed in vivo. Researchers use closed system models for their simplicity, high productivity, repeatability, controllability of the experimental conditions, less contamination, and cost-effective properties. The agar plate and microtiter biofilm models are two examples of a simple microbial culture model in closed system.

The open system

The open system can be described as a continuous culture cycle system. It enables the supply of a fresh culture medium and the removal of metabolites and spent culture liquid simultaneously. Hence, the concentration of bacteria and metabolic products remains constant.^[10] Moreover, the biofilm is able to stay in a stable state or in a dynamic balance.^[11] Nevertheless, the repeatability of experimental result is low because of the heterogeneity of the biofilm in the open system.

The open system simulates the in vivo environment better than the closed system, allowing better regulation of the biofilm growth rate and other variables. Common microbial culture models in the open system include the chemostat model, the flow cell biofilm model, the constant depth film fermenter model, the drip flow biofilm reactor, the multiple Sorbarod model, and the multiple artificial mouth model.

The multiple artificial mouth (MAM) is a computer-controlled, multiple station model. A MAM can accurately simulate an in vivo environment using computer-controlled facilities.^[12]

It has several microstations which are relatively independent to one another. Different experimental conditions can be applied simultaneously in different microstations.

Environmental variables can be easily controlled in the MAM, thus allowing analysis of the biofilm during its development without contaminating other samples. Acidity can be monitored using a pH electrode and a micro-reference electrode.^[6] These well-controlled conditions improve the standardization and flexibility of the MAM, therefore enhancing its ability to culture biofilms close to natural oral flora. Sissons et al. found that biofilms developed in this system exhibited metabolic and pH behaviour that resembled typical natural plaques.^[12] The MAM has been adopted in different studies, such as biodiversity of plaque, fluoride and phosphate assay^[13,14], plaque calcium level measurement^[14], and the generation of consortia using major plaque species¹⁵. The biofilm samples in this model were exposed to the same temperature and gas-phase fluctuation. The MAM aims to mimic the oral environment therefore saliva substitutes play an important role in the model. Approximate laminar flows are applied to simulate the situations in the oral cavity, instead of turbulent flow in chemostat.

Conclusions

The pre-calibration of a bioreactor system allows specification and refinement of its working parameters, thus contributing to accurate reproduction of the environmental conditions in the oral cavity. Nevertheless, the repeatability of the experimental result is rather limited, because of the heterogeneity of the biofilm in the open system. More data need to be collected regarding the resulting biofilms, formed in a dynamic open system.

References

1. Kolenbrander PE, London J. Adhere today, here tomorrow: oral bacterial adherence. J Bacteriol 1993; 175(11):3247–52.

2. Marsh PD, Bradshaw DJ. Dental plaque as a biofilm. J Ind Microbiol 1995; 15(3):169–75.

3. Foster JS, Kolenbrander PE. Development of a multispecies oral bacterial community in a saliva-conditioned flow cell. Appl Environ Microbiol 2004; 70(7):4340–8.

4. Yiru Yu, Zhao IS, Mei ML, et al. Dental biofilm and laboratory microbial culture models for cariology research. Dent J (Basel) 2017; 5(2):21.

5. Sissons CH. Artificial dental plaque biofilm model systems. Adv Dent Res 1997; 11:110–26.

6. Tang G, Yip HK, Cutress TW, et al. Artificial mouth model systems and their contribution to caries research: a review. J Dent 2003: 31:161–71.

7. Wimpenny JW. The validity of models. Adv Dent Res 1997; 11:150–9.

8. Coenye T, Nelis HJ. In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 2010; 83:89–105.

9. McBain AJ. Chapter 4: In vitro biofilm models: An overview. Adv Appl Microbiol 2009; 69:99–132.

10. Marsh PD. Host defences and microbial homeostasis: role of microbial interactions. J Dent Res 1989; 68:1567–75.

11. Sim CP, Dashper SG, Reynolds EC. Oral microbial biofilm models and their application to the testing of anticariogenic agents. J Dent 2016; 50:1–11.

12. Sissons CH, Cutress TW, Hoffman MP, et al. A multi-station dental plaque microcosm (artificial mouth) for the study of plaque growth, metabolism, pH, and mineralization. J Dent Res 1991; 70(11):1409–16.

13. Pearce EI, Sissons CH, Coleman M, et al. The effect of sucrose application frequency and basal nutrient conditions on the calcium and phosphate content of experimental dental plaque. Caries Res 2002; 36(2):87–92.

14. Wong L, Sissons CH. Human dental plaque microcosm biofilms: effect of nutrient variation on calcium phosphate deposition and growth. Arch Oral Biol 2007; 52(3):280–9.

15. Shu M, Wong L, Miller JH, et al. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol 2000; 45(1):27–40.