This research evaluated utilization of organic compounds and biomass accumulation in three different layered sand filter biofilm systems renovating a mixture of detergent and milk fat. Organic compounds were measured as chemical oxygen demand (COD) and biomass as ATP. A two-layer sand filter with coarse sand on the top and fine sand at the bottom; a three-layer sand filter with pea gravel on the top, coarse sand in the middle, and fine sand at the bottom; and a two-filters in-series sand filter biofilm system each received mixtures of butterfat and detergent. While the three-layer sand filter system had greater COD utilization capacity due to a longer filter run, the two-filters in-series sand filter system had greater COD utilization efficiency. The COD utilization was highly associated with the period of filter run to clogging. The decrease of COD utilization rate could lead to the clogging of sand filters. To maintain long filter runs without clogging, a COD loading rate of 2100 mg O(2)/m(2)/h was recommended for three-layer sand filters and 1800 mg O(2)/m(2)/h for two-layer sand filters for high fat content wastewater. In the two-layer sand filters and the three-layer sand filters, biomass did not seem to accumulate in any particular layer on a unit volume of sand. However, based on unit surface area, the coarse sand layer accumulated more biomass than the fine sand layer and the pea gravel layer.