It is important to control biofilm cohesiveness to optimize process performance. In this study, a membraneaerated biofilm reactor inoculated with activated sludge was used to grow mixed-culture biofilms of different ages and thicknesses. The cohesions, or cohesive energy levels per unit volume of biofilm, based on a reproducible method using atomic force microscopy (F. Ahimou, M. J. Semmens, P. J. Novak, and G. Haugstad, Appl. Environ. Microbiol. 73:2897-2904, 2007), were determined at different locations within the depths of the biofilms. In addition, the protein and polysaccharide concentrations within the biofilm depths, as well as the dissolved oxygen (DO) concentration profiles within the biofilms, were measured. It was found that biofilm cohesion increased with depth but not with age. Level of biofilm cohesive energy per unit volume was strongly correlated with biofilm polysaccharide concentration, which increased with depth in the membrane-aerated biofilm. In a 12-day-old biofilm, DO also increased with depth and may therefore be linked to polysaccharide production. In contrast, protein concentration was relatively constant within the biofilm and did not appear to influence cohesion.Biofilms are ubiquitous in nature, and they can be beneficial or troublesome, depending upon where and how they grow. There appears to be a consensus that the content of extracellular polymeric substances (EPS) is important in biofilm cohesion and biofilm adhesion to surfaces. For example, Klapper et al. (17) used a model based on polymer viscoelastic properties and suggested that the material properties of biofilm were largely determined by the EPS, implying that biofilm strength should indeed be linked to EPS quantity and composition. In addition, a recent study by Xavier et al. (35) proposed a kinetic model to describe biofilm detachment that was based on enzymatic disruption of the EPS matrix, thereby affecting biofilm cohesiveness.The EPS content of a biofilm can differ in quantity and character as a result of environmental factors. Numerous environmental factors have been reported to promote EPS production. These include high levels of oxygen (4), limited availability of nitrogen (15,22), desiccation (25), low temperature (16), low pH (28), and nutrient deprivation (20). Weiner et al. (34) described several roles and functions for EPS, including that of protection against environmental stress. In addition, Davies et al. (8) showed that activation of a gene (algC) for production of the exopolymer alginate was higher for Pseudomonas aeruginosa when attached to a Teflon mesh than for unattached P. aeruginosa. This suggests that organisms are able to respond to their environments and change EPS compositions and therefore their adhesion abilities, based on the surfaces to which they attach. Multivalent cations, such as those of calcium and magnesium, also probably play a role in the cohesiveness of microbial aggregates, as evaluated from studies of anaerobic sludge granules (12), activated sludge flocs (13), and biofilms ...