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The effects of four different filtration modes (i.e. relaxation, backwash, mixed and continuous) producing the same flux productivity (time average flux) on membrane fouling were investigated in membrane bioreactors (MBRs). The fouling behaviour was found to be strongly dependent on the applied instantaneous flux rather than the filtration modes themselves. The transmembrane pressure (TMP) obtained after 24 h of filtration was dominated by the fouling rates calculated within the first hour of the experiment. After the filtration experiments, the resulting fouling layers were fractionated by rinsing, backwashing and then chemical cleaning, with the foulant removal reflecting the strength of attachment to the membrane. An analysis of the three different fouling layers provided a unique insight into the composition (protein and carbohydrate) and spatial distribution of the particulate and soluble foulants. The upper fouling fraction consists of a porous, loosely bound cake layer with a similar composition to the biomass flocs. The intermediate fraction, which consists of equal parts of soluble molecular products (SMP) and biomass aggregates, features a higher concentration of carbohydrates and possibly plays a significant in the formation of consecutive cake layer. The lower fraction, representing the irreversible fouling fraction and predominantly consisting of SMP, features a relative higher concentration of strongly bound proteins. Whereas the lower and the intermediate fractions showed similar properties for all filtration modes, the upper fraction was influenced by the instantaneous flux.