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in an apparent gel network. Initial work shows that acetylcholinesterase (AChE) can be entrapped within the silicate particles. Preliminary enzyme kinetics work has shown that the entrapped AChE has a Vmax of 0.053 mol l−1 min−1 and an apparent Km of 0.002 M. These values are lower than those of the free enzyme and this is thought to be due to a combination of factors including the effect of the pH of the silicate surface environment and buffer used and the substrate concentration required by the entrapped enzyme (Miller et al., 2006). It is suggested that a higher substrate concentration is required for the entrapped enzymes in comparison to the free enzyme (Miller et al., 2006). This is thought to be due to competition of constrained enzyme within the silicate structures leading to a high local enzyme concentration and a faster consumption of the substrate, therefore not allowing the reaction to maintain a steady state (Miller et al., 2006). The future of this work will study how the enzymemodified-nanosilicate particles can be tethered to or formed on surfaces to support enzymes for synthesis and sensing. The reproducible deposition of self-assembling and self-organising inorganic structures that have the capability of interfacing with organic molecules could lead to the development of a whole range of materials in a vast number of industrially important areas. References Belton, D.J., Patwardhan, S.V., Perry, C.C., 2005. Spermine, spermidine and their analogues generate tailored silicas. J. Mater. Chem. 15, 4629–4638. Berne, C., Betancor, L., Luckarift, H.R., Spain, J.C., 2006. Application of a microfluidic reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene nitroreductase. Biomacromolecules 7, 2631–2636. Jin, R.H., Yuan, J.J., 2005. Simple Synthesis of Hierarchically Structured Silicas by Poly(ethyleneimine) Aggregates Pre-Organized by Media Modulation. Macromol. Chem. Phys. 206, 2160–2170. Kroger, N., Lorenz, S., Brunner, E., Sumper, M., 2002. Self-Assembly of Highly Phosphorylated Silaffins and Their Function in Biosilica Morphogenesis. Science 298, 584–586. Lopez, P.J., Gautier, C., Livage, J., Coradin, T., 2005. Mimicking biogenic silica nanostructures formation. Curr. Nanosci. 1, 73–83. Miller, S.A., Hong, E.D., Wright, D., 2006. Rapid and efficient enzyme encapsulation in a dendrimer silica nanocomposite. Macromol. Biosci. 6, 839–845. St¨ober, W., Fink, A., Bohn, E., 1968. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 26, 62–69.

doi:10.1016/j.jbiotec.2007.07.173 6. Degradation of toluene and xylene by Rhodococcus cells Carla C.C.R. de Carvalho, Vanessa Fatal ∗ , Sebasti˜ao S. Alves, M. Manuela R. da Fonseca Instituto Superior T´ecnico, IBB, Centro de Eng. Biol´ogica e Qu´ımica, Av. Rovisco Pais, 1049-001 Lisboa, Portugal Compounds such as benzene, toluene, ethylbenzene and xylene (BETX) are frequently released to the environment through e.g. petroleum, gasoline and diesel spills and leakage from storage containers. According to a study recently published, toluene and

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other non-halogenated compounds, rapidly infiltrate to aquifers (Tindall et al., 2005), urging the development of clean and fast degradation mechanisms. Toluene is a highly toxic compound and concentrations as low as 0.1% (v/v) kill most micro-organisms. Several Pseudomonas strains able to tolerate and grow in toluene concentrations higher than 50% (v/v) were isolated by Inoue and co-workers (1989, 1991), but these strains were unable to grow on toluene as sole carbon source. The two Rhodococcus strains, R. opacus PWD4 and R. erythropolis DCL14, used in the present study, were able to use toluene as sole carbon and energy source. The presence of xylene increased the rate of toluene consumption and the presence of toluene improved the rate of xylene degradation. The rate of consumption of both was significantly increased in aqueous:organic phase systems, in which the concentrations of toluene and xylene could be much higher than in single aqueous systems. The rate of toluene consumption by resting whole cells was dependent on the carbon source used for cell growth, on the solvent used as substrate reservoir and on the co-substrate used during the degradation experiments. The highest rates were achieved when toluene grown cells were used in aqueous:ndodecane systems using p-xylene as co-substrate. In fed-batch reactors, adaptation of R. erythropolis to the presence of toluene resulted in increased consumption rates along consecutive toluene additions. References Inoue, A., Yamamoto, M., Horikoshi, K., 1991. Pseudomonas putida which can grow in the presence of toluene. Appl. Environ. Microbiol. 57, 1560–1562. Tindall, J.A., Friedel, M.J., Szmajter, R.J., Cuffin, S.M., 2005. Part 1: Enhanced bioremediation of toluene in the unsaturated zone of a shallow unconfined aquifer. Water Air Soil Pollut. 168, 325–357.

doi:10.1016/j.jbiotec.2007.07.174 7. Biocatalysis for use in pharmaceutical manufacturing Francesco Molinari ∗ , Diego Romano, Raffaella Gandolfi, Roberto Gualandris, Nicola Ferrara Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale, Universit`a degli Studi di Milano, via Celoria 2, 20133 Milano, Italy Biotransformation techniques have evolved such that the synthetic chemist can utilize biocatalysts just as many other synthetic reagents are used. Natural microbial diversity and new techniques for further diversification offer an almost inexhaustible source of biocatalysts. The major goal of our research is the development of synthetically useful selective biotransformations using microbial enzymes by combining natural and non-natural conditions. The production of optically pure molecules to be used in pharmaceutical manufacturing has been studied. Different techniques have been exploited for improving the selectivity and/or productivity of the bioprocesses: medium manipulation (e.g. binding

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Abstracts / Journal of Biotechnology 131S (2007) S98–S121

agents or organic solvents), biocatalyst manipulation and reactor engineering (e.g. membrane reactors). Examples from our work will be presented with regards to these strategies, including different applications: • Chemo- and enantioselective oxidations with dehydrogenases from acetic acid bacteria • New reductions affording syn-diols • Selective modification of antibiotics (deacylation and demannosilation) by using hydrolases from actinomycetes. doi:10.1016/j.jbiotec.2007.07.175 8. Fermentation of glycerol to 1,3-propanediol by Klebsiella oxytoca NRTL B-199: Study of product inhibition Cristina Galdeano Villegas a,∗ , Victoria E. Santos a , Mercedes Zazo b , Jose Luis Garcia b , Felix Garcia-Ochoa a a

Facultad de Ciencias Quimicas, Universidad complutense de Madrid, Avda Complutense s/n, 28040 Madrid, Spain b Dpt. Microbiologia Molecular, Centro de Investigaciones Cient´ıficas, Madrid, Spain The recent development of a new polyester called poly(propylenterephtalate), a biodegradable polymer with unique physicochemical properties for the fibber industry has increased the attention towards the production of 1,3propanediol (1,3-PD), a product with this and other applications in cosmetics, foods, lubricants and medicines. Traditional chemical conversion of acrolein into 1,3-PD requires high temperature, high pressure and expensive catalysts. The microbial conversion of glycerol to 1,3-PD has recently received more attention because it is carried out using milder operational conditions than the chemical process and it does not generate toxic by-products. Moreover, the microbial process can use glycerol as substrate; therefore, it will be a very suitable compound when the biodiesel production had increased its production as consequence of the application of the European Directive 2003/30/CE, which imposes the growing use of biofuels. Glycerol is the main by-product obtained from biodiesel production around 10%. Glycerol can be naturally fermented into 1,3-PD by bacteria belonging to the genera Klebsiella, Clostridia, Citrobacter and Enterobacter under anaerobic or microaerobic conditions (Chen et al., 2003). In the biological production of 1,3-PD, a number of by-products (such as acetic acid, ethanol, 2,3-butanediol, etc.) may be simultaneously produced decreasing the yield in the main product, 1,3-PD. The highest yields are reported when bacteria of genera Klebsiella are employed (Huang et al., 2002). Several strains of Klebsiella sp. have been employed the microorganism used in this study has been Klebsiella oxytoca NRTL B-199, for which very few results have been published for the moment (Galdeano et al., in press). The aim of this work is to study the inhibition of K oxytoca growth by the main product of its fermentation, 1,3-PD, using glycerol as carbon source.

The medium composition used for the growth of K. oxytoca (and for the production of 1,3-PD, because it is an associated growth product) was determined and optimized by the Taguchi method in a previous work (Guang et al., 2007). It contains (per litre): 40 g glycerol, 0.15 g yeast extract, 0.5 g NH4 NO3 , 3.5 g K2 HPO4 , 1.5 g KH2 PO4 , 0.25 g Mg SO4 and 2 g NH4 Cl. K. oxytoca was incubated at 30 ◦ C, in an orbital shaker at 250 r.p.m. during 24 h. Samples were withdrawn from the flasks at one hour intervals. Cell growth was monitored as Optical Density at 600 nm. The concentration of glycerol, 1,3-PD and the other by-products were determined by HPLC using an Aminex HPX-87H column and a refractive index detector. The column temperature was 65 ◦ C and the detector temperature was 45 ◦ C. A solution of 5 mmol l−1 H2 SO4 was used as mobile phase at 0.8 mL min−1 flow rate. Inhibition experiments were carried out using different initial concentrations of 1,3-PD (0, 4, 8, 12, 16, 20 g/l). ␮(max) and the yield in 1,3-PD of the fermentation were calculated. Their values seem to be inversely proportional to the initial concentration of 1,3-PD. The presence of the 1,3-PD in the broth affects the glycerol fermentation more than the growth. These results show that the critical concentration of 1,3-PD contained in the broth is 16 g/L. References Chen, X., Xiu, Z., Wang, J., Zhang, D., Xu, P., 2003. Stoichiometric analysis and experimental investigation of glycerol bioconversion to 1,3-propanediol by Klebsiella pneumoniae under microaerobic conditions. Enzym. Microb. Technol. 33, 386–394. Galdeano, C., Zazo, M., Santos, V.E., Garcia, J.L., Garcia-Ochoa, F. Production of 1,3-Propanediol using Klebsiella oxytoca NRTL B-199 growing cells: medium composition optimization using Taguchi method. ECCE-6, in press. Guang, Y., Jiesheng, T., Jilun, L., 2007. Fermentation of 1,3-propanediol by a lactate deficient mutant of Klebsiella oxytoca under microaerobic conditions. Appl. Microbiol. Biotechnol. 73, 1017–1024. Huang, H., Gong, S.C., Tsao, G.T., 2002. Enhancement of oxygen transfer by pressure pulsation in aqueous glycerol fermentation. Appl. Biochem. Biotechnol. 98–100, 687–691.

doi:10.1016/j.jbiotec.2007.07.176 9. Isolation, cloning and synthetic use of the tHBP aldolase from Pseudomonas fluorescens N3 Silvia Ferrara a,∗ , Patrizia Di Gennaro a , Giuseppina Bestetti a , Fulvia Orsini b , Guido Sello b a

University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy b Department of Organic and Industrial Chemistry, University of Milano, Milan, Italy The use of biocatalytic processes for the industrial synthesis of chemicals has been attracting growing attention as a synthetic method that is both economically efficient and environmentally friendly. In this respect, enzymatic C-C bond formation represents a still scarcely developed reaction (Samland and Sprenger, 2006). We are now studying the potential of an aldolase to pre-