This study was to investigate effects of digested chicken manure filtrate (DCMF) supplementation on cells growth and lipid production in heterotrophic culture Chlorella protothecoides. Higher nitrogen DCMF favored cells growth but retarded lipid accumulation and vice versa for lower nitrogen DCMF. Accordingly, a two-step DCMF supplementation method of feeding higher nitrogen DCMF at cell growth phase and lower one at the stationary phase was adopted to enhance lipid production. Moreover, based on the phenomenon that feeding glucose at the stationary phase can further enhance lipid production, the strategies of DCMF supplementation coupled with adenosine triphosphate (ATP) regeneration from glucose feeding were developed. As a result, total lipid yield of 5.28 g/L was achieved after 200 h cultivation, increased by 74.8% and 21.1% respectively, compared to the control and the one without glucose addition. It was suggested that application of DCMF combined with glucose feeding as being feasible for lipid overproduction. (C) 2013 Elsevier Ltd. All rights reserved.