In this paper NaOH, Ca(OH)2, NH4OH, and H2O2 were applied to bagasse pith, to study their action on single cell protein production using a mixed culture of Cellulomonas flavigena and Xanthomonas sp. (strains CDBB-B-532 and ATCC-31920). The chemicals were sprayed under mild conditions as in previous work. Studies of pore size, lignin and phenolic content changes occurring on pith were performed since they correlate with carbohydrate utilization of lignocellulosic biomass. Fermentation data in decreasing order were: NaOH, Ca(OH)2, NH4OH, and H2O2. Material pretreated with NaOH and Ca(OH)2 showed increased cell growth as a function of time given by the logarithmic curve Y = A + BlnX, where Y represents cell growth, X is time and B is a constant. Decreased lignin content as a function of time was observed during pretreatment with calcium and sodium hydroxide, showing low delignification and relatively high amounts of phenolic compounds released, such as p-coumaric and ferulic acid. Scanning electron microscopy analysis showed that pith was mainly composed of microfibrils and parenchymatose cell wall. In addition, light microscopy presented swelling of vessels in pith pretreated with Ca(OH)2 and NaOH which was evident by the increased pore size of vessels in pith.