In this study, hydrogen partial pressure effects on formation of anaerobic biopellets were investigated. So, two-in-series UASB system with well defined biopellets was operated with acetic and propionic acid mixture( 1:1 as COD base) as substrate. As decrement of the hydrogen partial pressure in two-in-series UASB system, it could be observed a lot of biomass discharge from the first stage reactor, damaged biopellets in the second stage reactor, and dispersed sludges in settler. These were reflected in necessity of high hydrogen partial pressure as a key condition for pelletization process. Particularly, the evidence on growth of hydrogenotrophs and extracellular polymer excretions could not be found. And autolysis of microorganisms in declining growth or endogenous stage was observed in the second stage reactor with settler.