Biofilm cohesiveness measurement using a novel AFM methodology


F. Ahimou,1 M. J. Semmens,1 P. J. Novak,1 G. Haugstad2

1Department of Civil Engineering, University of Minnesota, 500 Pillsbury Dr. SE, and 2Characterization Facility, Institute of Technology, University of Minnesota, 100 Union Street SE, Minneapolis, Minnesota 55455

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 impacting 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 method to reproducibly measure, in situ, the cohesive energy of moist one 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 the cohesive energy increased with biofilm depth, from 0.10 ± 0.07 nJ/μm3 to 2.05 ± 0.62 nJ/μm3. This observation was reproducible, with four different biofilms showing the same behavior. The data suggested that the outer layers of the biofilm consist of mostly slime extracellular polymeric substances (EPS), whereas deeper layers contain EPS covalently bound to the microbial cell wall, which is significantly more difficult to remove. The cohesive energy also increased from 0.10 ± 0.07 nJ/μm3 to 1.98 ± 0.34 nJ/μm3 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 widely available off–the–shelf instrumentation. It could therefore be widely used to examine biofilm cohesion under a variety of conditions.