Joana Teodósio

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l−1) and a low (150 mg glucose l−1) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s−1 was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.

The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance

Aims:  This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions.

96-well microtiter plates for biofouling simulation in biomedical settings

Microtiter plates with 96 wells are routinely used in biofilm research mainly because they enable high-throughput assays. These platforms are used in a variety of conditions ranging from static to dynamic operation using different shaking frequencies and orbital diameters. The main goals of this work were to assess the influence of nutrient concentration and flow conditions on biofilm formation by Escherichia coli in microtiter plates and to define the operational conditions to be used in order to simulate relevant biomedical scenarios. Assays were performed in static mode and in incubators with distinct orbital diameters using different concentrations of glucose, peptone and yeast extract. Computational fluid dynamics (CFD) was used to simulate the flow inside the wells for shaking frequencies ranging from 50 to 200 rpm and orbital diameters from 25 to 100 mm. Higher glucose concentrations enhanced adhesion of E. coli in the first 24 h, but variation in peptone and yeast extract concentration had no significant impact on biofilm formation. Numerical simulations indicate that 96-well microtiter plates can be used to simulate a variety of biomedical scenarios if the operating conditions are carefully set.

Setup and Validation of Flow Cell Systems for Biofouling Simulation in Industrial Settings

A biofouling simulation system consisting of a flow cell and a recirculation tank was used. The fluid circulates at a flow rate of 350 L· h−1 in a semicircular flow cell with hydraulic diameter of 18.3 mm, corresponding to an average velocity of 0.275 m· s−1. Using computational fluid dynamics for flow simulation, an average wall shear stress of 0.4 Pa was predicted. The validity of the numerical simulations was visually confirmed by inorganic deposit formation (using kaolin particles) and also by direct observation of pathlines of tracer PVC particles using streak photography. Furthermore, the validity of chemostat assumptions was verified by residence time analysis. The system was used to assess the influence of the dilution rate on biofilm formation by Escherichia coli JM109(DE3). Two dilution rates of 0.013 and 0.0043 h−1 were tested and the results show that the planktonic cell concentration is increased at the lower dilution rate and that no significant changes were detected on the amount of biofilm formed in both conditions.

Flow cells as quasi-ideal systems for biofouling simulation of industrial piping systems

Semi-circular flow cells are often used to simulate the formation of biofilms in industrial pipes with circular section because their planar surface allows easy sampling using coupons. Computational fluid dynamics was used to assess whether the flow in pipe systems can be emulated by the semi-circular flow cells that are used to study biofilm formation. The results show that this is the case for Reynolds numbers (Re) ranging from 10 to 1000 and 3500 to 10,000. A correspondence involving the friction factor was obtained in order to correlate any semi-circular flow cell to any circular pipe for Re between 10 and 100,000. The semi-circular flow cell was then used to assess experimentally the effect of Reynolds number (Re = 4350 and 6720) on planktonic cell concentration and biofilm formation using Escherichia coli JM109 (DE3). Lower planktonic cell concentrations and thicker biofilms (>1.2 mm) were obtained with the lower Re.

The Effect of Plasmids and Other Biomolecules on the Effectiveness of Antibiofilm Agents

This chapter describes the impact of cell transformation with a recombinant plasmid and the effects of the presence of selected biomolecules (bovine serum albumin—BSA, alginate, yeast extract and humic acids) on biofilm resistance to quaternary ammonium compounds (QACs), which are often used in medical applications to prevent microbial contamination. Two case studies are presented, the first concerning cell transformation with recombinant plasmids and the second addressing potential interfering substances. In the first case study, the pET28 and pUC8 plasmids were used to transform Escherichia coli JM109(DE3), and biofilm formation, removal and antimicrobial susceptibility to the cationic biocide benzyldimethyldodecylammonium chloride (BDMDAC) were assessed. Plasmid-bearing cells formed biofilms with higher cell densities, whereas non-transformed cells had higher viabilities. It was found that biocide treatment was not efficient for biofilm removal and that the thickness of the biofilms formed by non-transformed cells is less affected by the treatment, a fact that can be associated with a higher protein content of the biofilm matrix. Despite being unsuccessful at removing the biofilms, BDMDAC was very effective at killing the cells since complete inactivation was attained for transformed and non-transformed strains. In the second case study, it was possible to conclude that BSA, alginate and yeast extract resulted in mild interferences in the antibacterial activity of benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB) against Bacillus cereus and Pseudomonas fluorescens. Humic acids have a severe impact on the activity of these QACs and can even trigger metabolic activation in some circumstances. These observations suggest that the presence of the tested biomolecules should be taken into account when using QACs as disinfection agents.