Biomolecular Engineering 17 (2001) 55–63 Bacillus subtilis cells immobilised in PVA-cryogels M. Szcze ˛sna-Antczak *, E. Galas Institute of Technical Biochemistry, Technical Uniersity of Lodz, 90 -924 Lodz, Stefanowskiego 4 /10, Poland Received 17 March 2000; received in revised form 23 August 2000; accepted 24 August 2000 Abstract Bacillus subtilis viable cells were immobilised in PVA-cryogel beads using the ‘freezing-thawing’ method in a two-phase (water – oil) system. Conditions providing both high thermal and mechanical stability and suitable porosity of the carrier were optimised. For monitoring and analysis of changes inside the biocatalyst beads, and for determination of diffusive properties of the carrier, an image analysis was applied. It was revealed that bacterial spores, sodium alginate and bacterial cellulose accelerated hardening of the cryogels and modified their porosity. Proteins (haemoglobin, azoalbumin, azocasein) penetrated beads of the cryogel. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Immobilisation; PVA-cryogels; B. subtilis ; Proteinases; Image analysis www.elsevier.com/locate/geneanabioeng 1. Introduction Modification of biotechnological processes, using im- mobilised biocatalysts, has recently gained attention of many biotechnologists. Application of immobilised en- zymes or whole cells is advantageous, because such biocatalysts display better operational stability [1,2], and frequently, higher efficiency of catalysis [3 – 5]. The aim of presented studies was to develop a method of immobilisation of viable Bacillus subtilis cells, possess- ing high physiological activity and able to produce extracellular subtilisin. The main problem, considering specific conditions of enzyme biosynthesis, was a choice of a mechanically and chemically stable carrier that allows diffusion of high molecular weight compounds (proteins). Adsorption of cells on porous carriers is the immobilisation procedure, which does not result in any additional diffusion barriers. However, its disadvantage are weak attractive forces between cells and the carrier that frequently bring about desorption of cells into the reaction milieu [6,7]. On the other hand, the entrapment of cells in usually applied natural polymers has some disadvantages, such as low mechanical stability, suscep- tibility to degradation in media containing enzymes (especially hydrolases) and considerable limitations for diffusion of large molecules. More chemically and me- chanically stable are synthetic polymers including poly- acrylamides, polymetacrylanes, polyuretans, etc. However, they are solidified in the presence of a biocat- alyst, which has to contact soluble, more or less toxic components of polymers. A highly promising alterna- tive to these carriers is a hard gel prepared from poly(vinyl alcohol) which is a (hot) water soluble syn- thetic polymer, forming stable, macroporous cryogels at low temperatures [8]. Poly(vinyl alcohol) is non-toxic to organisms and can be cheaply produced at industrial scale. The first enzyme entrapped in PVA-cryogels was -galactosidase [9], followed by cells of Citrobacter intermedius [10] and Zymomonas mobilis [11,12], applied as a source of L-tyrosine phenol liase and for conver- sion of glucose into ethanol. Recently the PVA-cryogels were applied for immobilisation of homoacetogenic bacteria for production of acetic acid from gaseous substrates [13,14], for transformation of hydrocortisone and progesterone [15], and as carriers for nitrifying microorganisms [16 – 19]. This paper presents results of studies on entrapment of viable B. subtilis cells producing proteolytic enzymes using poly(vinyl alcohol) cryogels. Abbreiations: ASM, textural coefficient of an angular second moment; CFU, colony forming units; DH, degree of hydrolysis; DP, degree of polymerization; PVA, poly(vinyl alcohol). * Corresponding author. Fax: +4-842-6313402. E-mail address: mirszcz@ck-sg.p.lodz.pl (M. Szcze ˛sna-Antczak). 1050-3862/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII:S1389-0344(00)00065-4