BACKGROUND: A mixture of primary and secondary municipal wastewater sludge (WWS) was tested in 40 day semi-continuous experiments as substrate for biogas production in a 2 MW agricultural biogas plant. To support industrial decision making during periods of substrate shortage seasonal extremes in sludge characteristics, effectiveness of ultrasonic and enzymatic pretreatments and their dual combinations were explored. RESULTS: Reanalysis of published data showed complex nonlinear relationship between specific ultrasound energy and disintegration degree of various WWS. The 51 bench-top reactor approach demonstrated a short adaptation period (15 days) to WWS and an increase in methane yields of 7.5%, 14.9% and 24.5% for the most effective ultrasound, hydrolytic and dual WWS pretreatments, respectively. The observed methane yields were notably lower from past batch experiments (Methane Yield Database V1.3). From the perspective of a 2 MW agricultural plant only enzymatic pretreatments of WWS were sustainable for full-scale deployment due to significantly lower costs of equipment installation, operation and maintenance. CONCLUSION: The approach described in this study allowed for sustainability analyses to be performed on site-specific basis, supported decision making on the most effective WWS pretreatment and provided the first full description of research protocols for effective assessment that can be extended to Standard Operating Procedures. (C) 2016 Society of Chemical Industry