In this work the application of hybrid materials, containing TEOS as source of SiO2 and k-carrageenan in different percentage, synthesized by the sol-gel method at room temperature was studied. They were used as matrices for entrapment of whole Bacillus sp. UG-5B cells, producers of thermostable nitrilase. The effect of the surface area and size and quantity of pores in the synthesized materials on the enzyme activity was evaluated. The process of biodegradation of different concentrations of toxic, potentially carcinogenic and mutagenic substrates by the obtained biocatalysts was investigated. The enzyme reaction takes place by the nitrilase pathway, catalysing nitrile hydrolysis directly to the corresponding carboxylic acid, forming ammonia. At batch experiments the influence of the substrate concentration of different nitriles was tested and 20 mM concentration was found most suitable. A two-step biodegradation process in a laboratory-scale column bioreactor of o-, m- and p-tolunitrile as a mixture was followed. After operation of the system for nine hours for the mixture of substrates at a flow rate of 45 mL h−1 and at 60°C, the overall conversion realized was above 90%, showing a good efficiency of the investigated process.