Immobilization of magnetic modified Flavobacterium ATCC 27551 using magnetic field and evaluation of the enzyme stability of immobilized bacteria Seyed Mortaza Robatjazi, Seyed Abbas Shojaosadati ⇑ , Rassoul Khalilzadeh, Ebrahim Vasheghani Farahani, Nooshin Balochi Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, P.O. Box 14115-114, Tehran, Iran article info Article history: Received 3 July 2011 Received in revised form 5 November 2011 Accepted 9 November 2011 Available online 23 November 2011 Keywords: Flavobacterium ATCC 27551 Immobilization Magnetic nanoparticles Magnetic modified bacteria abstract The magnetic modified Flavobacterium sp. was prepared by covalently binding carboxylate-modified magnetic nanoparticles, and also, ionic adsorption of magnetic Fe 3 O 4 nanoparticles on the cell surface. The magnetic modified bacteria were immobilized by both internal and external magnetic fields. The pH stability and inherent resistance of the enzyme activity of the immobilized bacteria under acidic and alkaline conditions were increased. Immobilization of the magnetic modified bacteria using an exter- nal magnetic field improved the enzyme thermal stability. The results revealed that immobilization of the magnetic modified bacteria by an external magnetic field keeps 50% of the enzyme activity after 23.4, 16.6 and 6 h of incubation at 55 °C for the covalently binding of magnetic nanoparticles, the ionic adsorp- tion of magnetic nanoparticles and the free cells, respectively. The results demonstrated the negative effect of various magnetic beads on the enzyme thermal stability of immobilized magnetic modified bac- teria using an internal magnetic field. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Immobilized cells are defined as ‘cells physically confined or localized in a certain defined region of space with retention of their catalytic activities, which can be used repeatedly and continu- ously’. This definition is applicable not only to cells, but also to sin- gle enzymes, multi-enzyme conjugating systems and cellular organelles that are involved in all types of biocatalysis (Flickinger and Drew, 1999). The use of immobilized whole microbial cells and/or organelles eliminates the often time-consuming and expen- sive steps involved in the isolation and purification of intracellular enzymes. It also enhances the stability of the enzyme by retaining its natural catalytic surroundings during immobilization and sub- sequent continuous operation (Babel and Kurniawan, 2004). Cell immobilization has been used in applications such as biotransfor- mation, hazardous material degradation and material production (Pan et al., 1997; Beshay et al., 2002; Najafpour et al., 2004; Amani and Amany, 2007; Kapoor and Kuhad, 2007). Microorganisms have been immobilized on a variety of matrices such as: agar, silica, cal- cium alginate, carrageenan and polyacrylamide (Nigam et al., 1998; Karandikar et al., 2006; Ha et al., 2009; Chibata et al., 1987; Petrov et al., 2007). Different methods such as physical entrapment in polymeric networks, attachment or adsorption to a carrier and microencapsulation have been used for cell immobi- lization. These methods have disadvantages such as the limited transport of substrates and the mechanical fragility of retaining materials in response to environmental changes, particularly in the presence of hydrodynamic shear. The unique properties of nanoparticles allow for their applica- tion in the modification of microbial cells. Superparamagnetism of magnetic nanoparticles (MNPs) is useful for applications requir- ing the manipulation of MNPs by a magnetic field. In most cases, the magnetic properties of the nanoparticles are generated by the presence of nano- or microparticles of magnetite (Fe 3 O 4 ) or maghemite (c-Fe 2 O 3 ). In some cases, ferrite particles or chromium dioxide particles can be used. Microbial cells can be magnetically modified by various methods including: non-specific attachment of MNPs, the binding or adsorption of maghemite particles or mag- netite particles on the cell surface, specific interactions with immunomagnetic nano and microparticles, covalent immobiliza- tion on magnetic carriers, the cross-linking of cells or isolated cell walls with a bifunctional reagent in the presence of magnetic par- ticles, and entrapment with magnetic particles into biocompatible polymers. Alternatively, the modification of cells can be performed by binding of paramagnetic cations to the acid groups on the cell surface. In many cases, the attached magnetic particles or ions have no negative effect on the viability and phenotype alteration of the modified cells (Safarik and Safarikova, 2007). Magnetically modified cells can be immobilized with an internal or external magnetic field. Moreover, cells bound to magnetic particles can 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.035 ⇑ Corresponding author. Fax: +98 21 82883381. E-mail address: shoja_sa@modares.ac.ir (S.A. Shojaosadati). Bioresource Technology 104 (2012) 6–11 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech