3D Printed Microfluidic Bioreactors Used for the Preferential Growth of Bacterial Biofilms through Dielectrophoresis

Abstract

A realistic modelling of the way biofilms form and evolve in time requests a dynamic approach. In this study, the proposed route uses continuous-flow bioreactors under controlled flow rates and temperature in the culture medium containing bacteria or fungi. 3D printed, Polylactic acid (PLA), flow-based bioreactors with integrated copper electrodes were used to investigate the effect of dielectrophoresis on the formation and growth of Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 13883 biofilms. Bacterial suspensions of 1McF turbidity have been prepared and circulated through the bioreactors. At the same time, a 30 V potential difference was applied on the system. The effect of the non-uniform electric field induced upon the bacterial cells was determined using quantitative methods, such as an adjusted microtiter plate technique, as well as spectral domain optical coherence tomography (SD-OCT) images. The morphology and the surface quality of the biofilms were investigated using Scanning Electron Microscopy (SEM) images. The results show that the different bacterial cells present a positive dielectrophoretic behaviour, with the preferential formation of biofilms in the high field gradient region.

Easy and Affordable: A New Method for the Studying of Bacterial Biofilm Formation

“…The SEM images for the biofilm formed inside the microfluidic devices ( Figure 7 ) presents more disorganized structures, with defined colonies, but no identifiable singular cells. This phenomenon could be explained by the experimental conditions, such as the sheer stress and flow rate present inside the microfluidic channels [ 24 ]. Another noticeable aspect is the arrangement of the colonies along the deposition lines for the Enterococcus and Klebsiella biofilms, with cervices between the filaments, and small connection areas between colonies.…”

“…As can be observed in the OCT images ( Figure 5 ), the deposition of biofilm inside the microfluidic devices is not uniform along the length of the channel, due to process parameters such as sheer stress and flow rate, which can lead to an irregular detachment of the biofilm. The thickness of the biofilm can vary both depending on the type of bacteria and its capacity to form biofilms, as well as on the investigated area (with thicker biofilm layers visible closer to the entrance and towards the center of the channels, and thinner layers towards the end of the channel [ 24 ].…”

“…The determination of total biofilm quantity was performed using the crystal violet technique previously described for a similar setting [ 24 ]. For the 3D printed plates, a sequence of staining with crystal violet 1% for 2 min was used: three consecutive rounds of washing-discoloration using alcohol.…”

Multispecies Bacterial Biofilms and Their Evaluation Using Bioreactors

“…Thus, STRs are suitable for use with shear-resistive microorganisms. Furthermore, Csapai et al [42] compared the effects of flow and electric field on biofilm formation in a microfluidic device, and found that biofilms grown under static conditions were well defined compared to biofilms subjected to the low flow rate of 0.1 mL/min (Reynold's number of 1.49 and flow velocity of 0.69 mm/s). The fluid flow conditions and flow parameters used in some biofilm studies are summarized in Table 2.…”