Abstract: Biofertilizers can be an alternative to chemical fertilizer as they increase sustainable soil fertility without causing pollution; however, their major problem is the poor survival of the free cells in the soil. A possible solution is the use of cells immobilized in biofilms; it provides a more suitable microenvironment for prolonged cell survival and allows the interaction of the bacterium and its metabolites with the plant. In this work, the planktonic Bacillus subtilis subsp. spizizenii showed a PGPR activity on Lactuca sativa, increasing the weight, 64 % the aerial part and 68 % the roots growth. This bacterium was able to produce a thick biofilm using glycerol as a sole carbon source. Different culture conditions were evaluated for biofilm production. The shear stress and the oxygenation during bacterial culture affected negatively the biofilm formation; a mechanically disrupted biofilm never recovered its integrity. The optimum temperature for biofilm production was between 30 ºC and 37 °C. The presence of different divalent cations salts affected the biofilm formation; 2 mM MgSO₄ and 1 mM FeSO₄ in static growth culture increased the biofilm production 36 % and 72 % respectively, and CoSO₄ and CuSO₄ affected negatively its formation. The immobilized cells had a PGPR effect; it showed a higher benefit as a biofertilizer than the planktonic form, producing an increment of 39 % of the aerial part and 59 % the roots growth.