A design of experiments was applied to evaluate different strategies to enhance the methane yield of macroalgae Gracilaria vermiculophylla. Biochemical Methane Potential (BMP) of G. vermiculophylla after physical pre-treatment (washing and maceration) reached 481 +/- 9 L CH4 kg (1) VS, corresponding to a methane yield of 79 +/- 2%. No significant effects were achieved in the BMP after thermochemical pretreatment, although the seaweeds solubilisation increased up to 44%. Co-digestion with glycerol or sewage sludge has proved to be effective for increasing the methane production. Addition of 2% glycerol ( w:w) increased the BMP by 18%, achieving almost complete methanation of the substrate (96 +/- 3%). Co-digestion of seaweed and secondary sludge (15:85%, TS/TS) increased the BMP by 25% (605 +/- 4 L CH4 kg(-1) VS) compared to the seaweed individual digestion. (C) 2014 Elsevier Ltd. All rights reserved.