The aim of this research was to investigate the nature and composition of organic substrate in two down-flow hanging sponge (DHS) systems using mixed fungal (FDHS) and bacterial (BDHS) cultures treatment for UASB effluent of tapioca starch wastewater, evaluated by COD fractionations and two material balances. The random type DHS reactors were operated as modular columns consisting of four identical segments connected vertically. Results of the wastewater characterization showed that carbonaceous fractions were varied on a function of DHS height. Two balances applied to experimental data were for chemical oxygen demand (COD) and nitrogen (N). Results of mass balance calculations can also be used to examine the process behavior of two DHS systems to improve the organic and nitrogen removal mechanisms.