Mainstream partial nitritation/anammox (PN/AMX) have attracted large attention in the last decade. The two-stage PN/AMX process has been pointed out as a more advantageous technology for mainstream anammox than one-stage systems. However, the process requires of an efficiency improvement by designing a new technology to remove the nitrate produced by anammox bacteria. This study has developed a new process by coupling anammox to heterotrophic denitrification in a single reactor unit by avoiding competition between both processes. The addition of acetate as an external C source enhanced heterotrophic denitrifying activity, allowing to reduce the nitrate produced by anammox bacteria, without hampering the anammox process. Further, a proper organic load management showed to be effective to face the drawbacks derived from previous mainstream treatment stages (i.e. undesired nitrate production), without compromising organic effluent quality. The combination of anammox and heterotrophic denitrifying in a single reactor unit was possible as anammox activity dominated within bottom sludge bed sections, while heterotrophic denitrification occurred within middle and upper sludge sections. Microbial diversity results of 16S rRNA gene-targeted sequencing analyses confirmed that anammox and heterotrophic denitrifiers communities occupied two differentiated sludge bed sections along the reactor. N2O emissions were lower when heterotrophic denitrification occurred.