Bioprocess Engineering 11 (1994) 209-212 9 Springer-Verlag 1994 Comparative analysis catalysts for gluconic D. Subba Rao, T. Panda of different whole acid fermentation cell immobilized Aspergillus niger using pretreated cane molasses Abstract To compare the efficiency of various whole cell immobilization techniques for the production of gluconic acid by Aspergillus niger were investigated using potassium ferrocyanide-treated cane molasses as the substrate. The techniques followed were: (1) Calcium alginate entrapment, (z) cross-linking with glutaraldehyde after cell permeabilization with (a) acetone, (b) toluene and (c) isopropanol and (3) development of granular catalyst. A comparative analysis of yield has revealed that calcium alginate entrapment was the most suitable technique as it had given the maximum product yield (o.4o g gluconic acid/g total reducing sugar supplied). The properties of immobilized A.niger in sodium alginate gel have been thoroughly investigated and compared with those of free cells under most suitable conditions of fermentation. 1 Introduction Gluconic acid and its derivatives have a wide use in food and pharmaceutical industries. The manufacture of gluconic acid is mainly by batch fermentation processes using Aspergillus sp. and Penicillium sp. besides Gluconobacter sp. and Zymomonas mobilis [1]. The existing fermentation processes are fairly efficient but a continuous process on the basis of an immobilized biocatalyst seems to have more potential. Immobilization of microbial cells is often beneficial when they are to be used in biotechnological processes [z]. The advantages of immobilized cells over free cells are: Reuse of the biocatalyst; higher conversion rate; reduced susceptibility to contamination and easy separation from reaction products. In the present study, investigations have been made to compare the efficiency of various reported whole cell immobilization techniques for the Received 6 October i993 T. Panda D. Subba Rao Division of BiochemicalEngineering, Department of Chemical Engineering, Indian Institute of Technology, Madras 6oo o36, India Correspondence to: T. Panda The Head, RSIC, IIT Madras is duly acknowledged for providing facility to analyse the metal ions by ICPS. Authors also thank Mr. S. Venkatesan for excellent typing of the manuscript. production of gluconic acid by Aspergillus niger using treated cane molasses as the substrate. Also, the alginate entrapment technique has been optimized as it was found to be most suitable and its efficiency is compared to that of free cells. 2 Materials and methods 2.1 Organism Aspergillus niger, NCIM 548, obtained from the National Chemical Laboratory, Pune, India, was used throughout this study. 2.2 Culture maintenance and growth conditions The culture was maintained on a potato-dextrose-agar medium having in kg/m3: Glucose - 25, potato - zoo and agar- zo [3]. For the growth of rnycelium, the following germination medium was used (kg/m~): Glucose - 30, (NH4)zHPO4 - 4.2, urea - 1.1, KHEPO 4 - z.o and MgSO4.7H20 - 1.7. The pH of the medium was adjusted to 6.5 with 5 M NaOH before inoculation. 6% (v/v) spores suspension (3 • lo7 spores/cm3) was used as the inocnlum. The spores were grown for z4 h at 3o ~ in 250 cm 3 Erlenmeyer flasks containing 5o cm 3 of germination medium on a rotary shaker at 16o rev/min. 2.3 Cell harvesting Cells were separated from the germination medium by centrifugation and washed thoroughly with O.Ol M citrate buffer (pH 6.5). From the residue, 0.39 g dry weight equivalent of cells was used in each of the immobilization techniques. 2.4 Calcium alginate entrapment Cells were mixed with 30 kg/m 3 sodium alginate solution and the resulting mixture was extruded through a nozzle (i.d. 3 ram) into a suitable CaC12 solution as per the requirement of the experiment. After hardening for 2 h at 30 ~ in the stirred CaC12 solution, the beads were washed thoroughly with o.ol M citrate buffer (pH 6.5) and stored in the same buffer at 4 ~ until further usage. 209