Streptavidin has been exploited as affinity tag to isolate protein in biotinilated columns. Recombinant Pichia pastoris KM71/Stp strains containing the streptavidin gene were fed-batch cultured generating 150 g L^-1 biomass. This biomass was achieved with a simpler and shorter process that has never been reported. The yeast was pre-cultured into 50 mL minimum medium with glycerol as only carbon source at 25 ºC and 250 rpm. After 12 hours incubation, the culture was transferred to a bioreactor with 200 mL of the same initial A₆₀₀ 0,2. After 24 hours incubation the culture was fed-batch with glycerol and basal salts medium to reach 400 mL. Aeration a porous stone as oxygen disperser in the air injection system. During the shift from biomass growth phase, to induction phase methanol feeding part of the cell (200mL) was immobilized in calcium alginate beads. Streptavidin production was evaluated in two systems: immobilized cells and free cells extract. The free cell system outcome was 4,0g L^-1 and the immobilized system was 25% lower streptavidin, active biologically, 96 hours methanol feeding. The biomass re-utilization was evaluated and the streptavidin production decreased. The streptavidin, released in the culture medium was 3,2 e 0,87 g L^-1 respectively in the free cells system. The immobilized system showed values 50 % and 80% related to free system respectively in second and third utilization. The recombinant P. pastoris biomass immobilization and reutilization have not been reported yet. This streptavidin production system has demonstrated to be a powerful technique.