Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 80:502–510 (2005) DOI: 10.1002/jctb.1204 Production of Bacillus thuringiensis-based biopesticides in batch and fed batch cultures using wastewater sludge as a raw material A Yezza, 1 RD Tyagi, 1∗ JR Val ´ ero 1,2 and RY Surampalli 3 1 INRS Eau, Terre et Environnement, 2800 Rue Einstein, CP 7500, Sainte-Foy, Qu ´ ebec, Canada, G1V 4C7 2 Centre Canadien des For ˆ ets, Centre Foresterie des Laurentides 1055 du PEPS, PO Box 3800, Sainte-Foy, Qu ´ ebec Canada, G1V 4C7 3 US Environmental Protection Agency, PO Box 17-2141, Kansas City, USA, KS 66117 Abstract: Bacillus thuringiensis subsp kurstaki was grown in batch and fed batch cultures using wastewater sludge as a raw material. A simple fed batch strategy based on dissolved oxygen measurement during the fermentation cycle was developed in this work. It was established that while shifting the process strategy from batch to fed batch, the maximum spore concentration was increased from 5.62 × 10 8 to 8.6 × 10 8 colony forming units per cm 3 and resulted in an increase of entomocidal activity from 13 × 10 9 to 18 × 10 9 spruce budworm potency units per dm 3 . Higher entomotoxicity was recorded at low spore concentration using wastewater sludge as a raw material whereas low entomotoxicity was reported at high spore concentration in synthetic medium.  2005 Society of Chemical Industry Keywords: Bacillus thuringiensis; wastewater sludge; fed batch culture; entomotoxicity; protease activity; sporulation 1 INTRODUCTION High protein (toxin) productivity or entomocidal activ- ity is the goal for the process optimization of Bacillus thuringiensis (Bt) fermentation using synthetic medium or natural complex medium (such as wastewater sludge) as raw materials. In a batch culture, the opti- mization of different fermentation parameters, namely temperature, pH, agitation and aeration, volume and age of inoculum, sludge suspended solids concentra- tion, C/N ratio and various pre-treatments methods of sludge undeniably allowed higher entomotoxicity (Tx) to be achieved in the final product. 1–6 In fact, all our previous studies on Bt fermentation using sludge as a raw material have been done in conventional batch process. The principal disadvantages of a batch process are the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermenter vessel, the cleaning, calibration, and sterilization, and process re-starts. However, the cell and spore concentration and hence the Tx value can be further increased by adopting various operational process strategies. Fed batch fermentation provides a valuable tool in order to increase productivity and a concomi- tant decrease in product manufacturing cost. 7–10 The outcome of fed batch culture depends on many variables such as fermentation time, feed rate profile, availability of nutrients in the medium, oxygen concentration and pH profile. 11 The main issue of such systems is to maintain an appro- priate feeding strategy to prevent overfeeding or underfeeding. 12 – 15 The fed batch operational strategy has been successfully studied to augment Bt cell and spore counts and the process productivity using conventional synthetic medium. Kuppusamy and Balaraman 14 established that high cell density of Bt H-14 could be achieved without any negative effect on sporulation and endotoxin expression by adopting a simple fed batch control based only on glucose limitation. The concentration of Bt cells could be improved from 6 g dry weight (dw) dm −3 in a batch system to well over 50g dw dm −3 in a well-designed fed batch operation in a laboratory fermenter using synthetic medium. 16 Kang et al 17 obtained a spore concentration of 1.25 × 10 10 spores cm −3 by employing intermittent fed batch culture in modified glucose–yeast extract–salt (GYS) medium. Using a fed batch strategy based on motile intensity, Chen et al 18 showed that the maximum cell concentration increased up to 50% compared with the batch culture. However, all these studies were carried out using synthetic medium and toxin yield or Tx ∗ Correspondence to: RD Tyagi, INRS Eau, Terre et Environnement, 2800 Rue Einstein, CP 7500, Sainte-Foy, Qu ´ ebec, Canada, G1V 4C7 E-mail: tyagi@inrs-ete.uquebec.ca Contract/grant sponsor: Natural Sciences and Engineering Research Council of Canada; contract/grant number: A4984 Contract/grant sponsor: Natural Sciences and Engineering Research Council of Canada; contract/grant number: STP235071 (Received 20 April 2004; revised version received 13 September 2004; accepted 20 September 2004) Published online 21 February 2005  2005 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2005/$30.00 502