Background

Biofilm is the most prevalent growth mode of microbial organisms in nature, industry, and clinical habitats; it is commonly recognized as bacterial communities embedded in the self-generated matrix of extracellular polymeric substances (EPS) (Flemming et al., 2016). The spatial organization of a biofilm aggregate is highly heterogeneous and has diverse metabolic activities, rendering the biofilm robust and tolerant to numerous types of environmental stress (Van Dyck et al., 2021). Hence, biofilms are promising in industry to ferment for nutrient conversion, degrade hazardous compounds for bioremediation, and generate electricity in microbial fuel cells (Coenye and Nelis, 2010). Conversely, biofilms also raise public concerns such as biofouling and biofilm-associated infections. Biofilms are the leading cause of chronic wound infections and infections on biomedical devices, such as cardiac valves, tracheal tubes, and catheters (Del Pozo et al., 2018). An improved understanding and characterization of biofilm formation, dispersion, and activities will shed light on biofilm control strategies.

A typical biofilm life cycle usually involves 5 stages: initial reversible attachment of bacteria, stable and irreversible attachment, maturation, dispersion, and dispersed free-living bacteria (Martin et al., 2021). The biofilm communities show active social behaviors and interaction with environmental factors, which in return regulate the cellular metabolism of biofilms. Numerous methodologies and devices have been documented for the investigation of biofilm morphology and development in vitro, commonly categorized as static biofilm assays and continuous-flow biofilm systems. The static biofilm assays - such as those in microplates and on air-liquid interface coverslips or colony biofilm assays and Kadouri drip-fed biofilm assays - are prevalently applied to the screening of early events in biofilm formation (Merritt et al., 2005). The static biofilm assays are high-throughput and easily executed with common laboratory equipment; however, nutrient supply in static biofilm assays is limited with respect to developing mature biofilm communities that mimic nature.

Given the fact that biofilms are frequently observed under fluidic flow conditions in nature and engineered systems, nutrient availability, physical transport, and hydrodynamic shear forces significantly impact biofilm formation and metabolism (Mattei et al., 2018). To address the question of how biofilm responds to environmental stresses and communicating signals, it is important to create a standardized and reproducible protocol for cultivating in vitro biofilms in flow systems mimicking the in vivo fluid habitats (Cowle et al., 2020). The development of flow biofilm reactors has been advanced by emerging microfluidics manufacturers and designs, with the most common flow systems including drip-flow systems (Gonzalez et al., 2014), tubular reactors (Winn et al., 2014), planar flow-cell (Zhang et al., 2011), and 3-channel flow-cell (Sternberg and Tolker-Nielsen, 2006; Pamp et al., 2009). Further modifications, such as segmented flow cells (Karande et al., 2014), gradient-generator flow cells (Zhang et al., 2019), and flow-velocity microfluidic flow cells (Liu et al., 2019), are specifically modified and designed to meet specific research purposes.

In this protocol, we addressed three commonly applied flow-system setups for different research purposes with Pseudomonas aeruginosa PAO1 and Shewanella oneidensis MR-1 as model organisms. The tubing biofilm reactor mimics the biofilm habitats in pipelines, tracheal tubes, catheters, etc., allowing biofilm harvest with adequate biomass. The 3-channel flow-cell system is designed for the non-invasive spatiotemporal observation of biofilm morphologies with a continuous and steady nutrient supply. The recycling biofilm reactor enables the study of biofilm metabolic responses toward its bioremediation metabolites and/or environmental chemicals (Sternberg and Tolker-Nielsen, 2006; Weiss Nielsen et al., 2011; Pamp et al., 2019). Overall, the standardized flow systems promise productive and reproducible biofilm observation and quantitation, which can be further modified according to the specific research project.