This study proposes an approach to understand biofilm formation in polyphasic anaerobic digestion systems. The objective was to better control biofilm formation in order to reduce the time of colonization during the start-up phase of an anaerobic high-rate biofilm reactor. A three-phase (liquid–gas–solid) inverse turbulent bed reactor (ITBR), with low-density particles used as biomass carrier, was started by stepwise increase of the organic loading rate (OLR) from 0.5 to 20 gCOD L-1 day-1 while maintaining a COD removal rate of 80%. The reactor was operated at a short and constant hydraulic retention time to favor biofilm accumulation. Biofilm formation and reactor performance have been followed for 90 days. In the initial phase, a COD removal of more than 90% was observed, together with continuous biofilm growth. Thereafter, at a loading rate of 6 gCOD L-1 day-1, the reactor efficiency suddenly deteriorated, volatile fatty acids (VFA) accumulated in the effluent and the specific methanogenic activity of the biofilm dropped down. This phenomenon was due to a micronutrient limitation, especially cobalt and nickel. After complementation with a mineral solution, optimal methanogenic activity and COD removal were restored. The nutrient supplementation was required to keep on increasing the OLR until the reactor reached a volumetric organic loading rate of 20 gCOD L-1 day-1 with more than 90% of carbon removal efficiency.