Anaerobic digestion (AD) of complex substrates is a multi-step process, which is kinetically controlled by an individual rate-limiting step. A methodology for determining the rate-limiting step during AD of complex substrates was developed by supplementation of metabolic intermediates from each digestion step with dairy manure as an emblematic complex substrate. This method elucidated that hydrolysis of dairy manure was the rate-limiting step when normal anaerobic sludge was used as inoculum. Furthermore, the concept and effect of microbial community ratio was introduced by manipulating two different inocula, i.e. normal anaerobic sludge and heated anaerobic sludge, so that varying ratios (r) of hydrolytic and methanogenic bacteria could be studied. Results revealed that the rate-limiting step changed with the variation of r. For dairy manure, results indicated a critical ratio r* = 24 between hydrolytic bacteria and methanogens, whereby as r decreased or exceeded from this value, hydrolysis or methanogenesis limited the AD process, respectively. (C) 2013 Elsevier Ltd. All rights reserved.