Biofilm Cohesiveness Measurement Using a Novel Atomic Force Microscopy Methodology

Abstract

Biofilms can be undesirable, as in those covering medical implants, and beneficial, such as when they are used for waste treatment. Because cohesive strength is a primary factor affecting the balance between growth and detachment, its quantification is essential in understanding, predicting, and modeling biofilm development. In this study, we developed a novel atomic force microscopy (AFM) method for reproducibly measuring, in situ, the cohesive energy levels of moist 1-day biofilms. The biofilm was grown from an undefined mixed culture taken from activated sludge. The volume of biofilm displaced and the corresponding frictional energy dissipated were determined as a function of biofilm depth, resulting in the calculation of the cohesive energy. Our results showed that cohesive energy increased with biofilm depth, from 0.10 ؎ 0.07 nJ/m 3 to 2.05 ؎ 0.62 nJ/m 3 . This observation was reproducible, with four different biofilms showing the same behavior. Cohesive energy also increased from 0.10 ؎ 0.07 nJ/m 3 to 1.98 ؎ 0.34 nJ/m 3 when calcium (10 mM) was added to the reactor during biofilm cultivation. These results agree with previous reports on calcium increasing the cohesiveness of biofilms. This AFM-based technique can be performed with available off-the-shelf instrumentation. It could therefore be widely used to examine biofilm cohesion under a variety of conditions.It is essential to understand biofilm stability to both encourage biofilm maintenance in some applications, such as waste treatment, and effectively remove undesired biofilm in others, as in biofilms covering medical implants. Biofilm detachment is one of the critical factors that balance growth and plays a role in the development of biofilm spatial heterogeneity. While factors responsible for biofilm growth are well studied (16,29,39,42,43), those controlling the detachment process are not clearly understood (28,36,38). As a consequence, a good understanding of the relationships between operating conditions and biofilm cohesion is lacking. The cohesive strength of the biofilm is influenced by extracellular polymeric substances (EPS) and specific compounds, such as calcium, which fill the space between microbial cells and bind cells together (23,30). Understanding the cohesive interactions in the biofilm matrix under a variety of conditions could lead to the design of new strategies for controlling biofilm development based on disrupting or protecting the matrix holding the biofilm together.Because cohesive strength is a primary factor affecting biofilm sloughing, its quantification is essential in understanding detachment. A few methods based on the use of custom devices have been proposed to investigate biofilm cohesive strength. Poppele and Hozalski (31) measured the tensile strength levels of biofilms from activated sludge by using a micromechanical device based on the deflection of a glass micropipette separating a microbial aggregate held by suction. Körstgens et al. (22) used a uniaxial compression measurement device to determine the yield st...

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