BACKGROUND: 11 alpha-hydroxyandrosta-1,4-diene-3,17-dione (11 alpha-OH-ADD) serves as an important steroid intermediate for many hormone pharmaceuticals. However, the poor aqueous solubility of the beta-sitosterol which is the substrate hinders its productivity efficiency. This paper reports a new method of biotransformation of beta-sitosterol to 11a-OH-ADD by mixed cultivation of Mycobacterium MF006 and Aspergillus ochraceus MF007 with organic silica hollow spheres (OSHS) to improve the dispersibility of the substrate based on its excellent adsorption property. The effect of co-cultivation method and OSHS on the biotransformation were evaluated. RESULTS: Results obtained by scanning electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis indicated that the adsorption complexes were formed with beta-sitosterol and OSHS, which increased the opportunity for interaction between microorganisms and beta-sitosterol. Using adsorption complexes as substrates, the conversion efficiency increased to 87.11% and the yield of 11 alpha-OH-ADD enhanced one and a half times through the synergy of these two microorganisms. CONCLUSION:This study is the first to demonstrate that OSHS can increase the solubility of beta-sitosterol and the mixed cultivation of microorganisms is able to enhance production and shorten duration, which provides a new direction for enhancing the biotransformation efficiency of steroids. (C) 2016 Society of Chemical Industry