Due to the large-scale production and wide applications, many nanoparticles (NPs) enter wastewater treatment plants and accumulate in activated sludge. It is reported that titanium dioxide (TiO2) NPs show severe damage to many model microbes. However, it is still unknown whether the long-term (e. g., 100 d) presence of TiO2 NPs would affect the performance of sludge fermentation. In this study, long-term exposure experiments (105 d) were conducted to investigate the potential risk of TiO2 NPs to sludge fermentation system. It is found that the presence of environmentally relevant [6 mg. (g TSS)(-1)] and higher [150 mg. (g TSS)(-1)] concentrations of TiO2 NPs does not affect methane production from sludge fermentation. The analysis of fluorescence in situ hybridization indicates that these concentrations of TiO2 NPs present marginal influences on abundances of bacteria and methanogenic archaea in sludge fermentation system. The viability of sludge microorganisms and activities of key enzymes related to methane production such as protease, acetate kinase, and coenzyme F-420 are unchanged by the long-term presence of 6 and 150 mg. (g TSS)(-1) of TiO2 NPs. Further investigations reveal that the insolubility of NPs and the protection role of sludge extracellular polymeric substances are the main reasons for the marginal influence of TiO2 NPs on sludge fermentation. (C) 2014 The Chemical Industry and Engineering Society of China, and Chemical Industry Press. All rights reserved.