A large fraction of the organic substrate in municipal wastewater is particulate. Prior to uptake, particles have to be degraded through potentially a range of intermediates. However, research on intermediate dynamics during particle hydrolysis is limited. In this paper, batch experiments on flocculated and dispersed biomass microcosms using starch as particulate substrate are reported. Overall hydrolysis rate was not significantly different between the two systems. Particle colonization, and increased particle porosity in combination with particle breakup, led to increased substrate availability over time. Particle breakup was more important for flocculated biomass, while increased particle porosity and particle colonization played a larger role for dispersed biomass. During particle degradation intermediates were formed; however, all intermediate polymer sizes were not formed to the same extent. This can be explained by non-random enzymatic degradation, where some products are preferred over others. Intermediates' dynamics also depend on the biomass structure, and in a floc-based system, diffusion limitations allow glucose to accumulate in the system.