Different activated sludges were used for developing an inoculum able to degrade hydrogen sulphide in a pilot scale biofiltration plant using two different support materials: sugarcane bagasse and this bagasse mixed with pumice stone. Adapting and selecting microbial species which degrade hydrogen sulphide (H2S) was aided by adding nutrients plus a specific substrate to the activated sludge. Population variation was monitored within the different trophic groups in the biofiter medium during pilot scale plant operation, a general trend towards sulphur-oxidising bacteria (SOB) growth being observed as was a decrease in heterotrophic bacteria, molds and yeasts. The activated sludge which showed the highest substrate degradation speed was selected for standardising inoculum preparation; the different nutritional mediums were evaluated during this process. Measuring some variables for controlling the process led to choosing the pH for determining the proper point of inoculum adaptation for this specific substrate. The inoculation procedure and support characteristics in terms of establishing and developing the microbial species increased biofilter removal efficiency by up to 99% from start-up. Key words: biofilter, activated sludge, adapted microorganisms, sulphur-oxidising bacteria, respirometry. Este articulo es el resultado de un proyecto cofinanciado por Colciencias y desarrollado por un grupo de investigadores vinculados al proyecto a traves de las entidades Corporacion