We studied the influence of bioavailability of organic matter on membrane fouling layer development by comparing the filtration performance of two feed waters (wetland water and graywater). Dissolved organic carbon (DOC) concentration, size exclusion chromatography (SEC), and fluorescence excitation-emission matrix (FEEM) were used to characterize the bioavailability of organic matter in these water samples during the nanofiltration process. The wetland sample contained a high proportion of humic acid-and fulvic acid-like matter with low bioavailability, whereas the graywater sample comprised substantial amounts of aromatic proteins and microbial byproduct-like matter with high bioavailability. In addition, the molecular size distribution revealed that the wetland sample contained a large portion of recalcitrant organic matter, whereas the graywater sample contained easily bioavailable organic matter. After the filtration experiment, the DOC of the wetland sample decreased to 4.8 mgC/L, whereas the graywater sample resulted in a lower DOC concentration of 3.4 mgC/L. Optical coherence tomography (OCT) illustrated real-time variations in the fouling layer morphology, providing both 2D and 3D images. In addition, confocal laser scanning microscopy (CLSM) quantified the bacterial volume in the fouling layer. The wetland sample yielded a bacterial volume of 11.8 mu m(3)/mu m(2) from a total fouling volume of 103 mu m(3)/mu m(2), whereas the graywater sample yielded a bacterial volume of 53.2 mu m(3)/mu m(2) from a total fouling layer volume of 134 mu m(3)/mu m(2). Fitting of the two-phase Monod model to the fouling layer growth on the membrane resulted in lower-yield coefficients (i.e., the volumes produced per unit amount of substrate, Yxs) of 7.46 and 27.95 mu m(3)/mu m(2) in wetland water and higher-yield coefficients of 13.17 and 47.53 mu m(3)/mu m(2) in the graywater at first and second phase, respectively. This study addresses the quantitative evaluation of the organic matter bioavailability in terms of membrane fouling using OCT images and a two-phase Monod model.