# A dual-loop chemostat to investigate multi-species biofilms on implant surfaces under adjustable flow conditions

## TL;DR

A new reactor system allows studying multi-species biofilms on implant surfaces under controlled flow conditions, revealing how flow affects biofilm structure and composition.

## Contribution

A modular dual-loop reactor was developed to independently control nutrient availability and flow rate for biofilm studies.

## Key findings

- Biofilm mass, robustness, and species distribution differ significantly under static and dynamic growth conditions.
- At high flow rates, late colonizers like Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans had lower abundance.
- The system enables systematic testing of surface materials and antimicrobial coatings under defined flow regimes.

## Abstract

Natural biofilms are typically composed of a mix of different microbial species and are often exposed to strong shear forces resulting from liquid flow. Simple biofilm models that attempt to study biofilms are based on a single species and on static growth conditions. To overcome these limitations, we developed a modular dual-loop reactor that decouples bacterial cultivation from hydrodynamic exposure, enabling independent control of nutrient availability (and thus, cell density) and flow rate (and thus, shear stress). Importantly, the system allows for testing different surface materials in a systematic manner. To validate our setup, we used a community of six keystone members of oral biofilms in conjunction with titanium materials of defined roughness that mimic dental implant surfaces. We found that biofilm mass, robustness, and species distribution not only differ significantly between static and dynamic growth conditions, but also vary strongly with different flow velocities. The biofilms formed under flow could be separated into two fractions, one that washed away very easily, and a more robust, basal layer. At low shear forces, overall biofilm mass was the highest, but at the expense of biofilm robustness. At medium shear forces, the robust fraction of the biofilm had the highest relative content of extracellular matrix. At the highest flow rates, the biofilm mass was low, but late colonizers (represented by the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans) had the lowest relative abundance. This is in accordance with the concept that high flow of saliva reduces the risk of oral disease. Future applications of our system will include the systematic testing of antimicrobial coatings or surface design effects under defined flow regimes, opening the path toward better medical implants.