A new immobilization process using chitosan was developed for use in L-citrulline production. In this process, a cell suspension of Enterococcus faecalis and chitosan solution were mixed and cross-linked with glutaraldehyde. After treatment with a solution of saturated sodium bicarbonate, stirring, and washing, the immobilized cells were filtered. A fermentation broth of E. faecalis cells containing arginine deiminase was used for immobilization without the use of a cell-separation step and then compared with immobilized separated cells in a 1.0-L batch conversion. Cells immobilized with fermentation broth showed increased operational stability and were used in scale-up procedures. On an industrial scale, 210 kg of cells immobilized from 2000 L of fermentation broth was used for a 2000-L reaction. After five batches, 1000 kg of L-arginine was converted to 974.6 kg of L-citrulline, yielding 733.5 kg of the final product. The results demonstrate the feasibility of immobilization using chitosan on an industrial scale. (C) 2015 Elsevier Ltd. All rights reserved.