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A novel packed-bed bioreactor, operating under isothermal and non-isothermal conditions, has been constructed. The core of the apparatus consisted in a polypropylene ring filled with beta-galactosidase immobilized on beads of polyacrylic acid, grafted with dimethylaminoethyl methacrylate. Phenylendiamine and glutaraldehyde were used as spacer and coupling agent, respectively. Two lateral nylon membranes held the enzyme beads into the ring and allowed the occurrence of the process of thermodialysis when the bioreactor was operating under non-isothermal conditions. Comparison of the enzyme activity under isothermal and non-isothermal conditions has shown that in the presence of temperature gradients the rate of lactose hydrolysis was increased, with a reduction of the apparent Km value. Under non-isothermal conditions the percentage increases of enzyme activity were found to decrease with the increase of the substrate concentration. The results have been explained within the frame of reference of the process of thermodialysis.

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.

The bioremediation of waters polluted by Bisphenol A, taken as a model for endocrine disruptors, has been pursued by means of catalytic membranes in bioreactors operating under isothermal and non-isothermal conditions. Laccase from Trametes versicolor was immobilized on nylon membranes grafted with Glycidyl Methacrylate and using Phenylendiamine as spacer. The behaviour of the catalytic membrane was studied as a function of BPA concentration. Affinities of immobilized laccase towards BPA were found to increase with average temperature and under nonisothermal conditions. Percentage increases of enzyme activity, proportional to the applied temperature differences, were found to decrease with the increase of BPA concentrations. Interestingly, the highest levels of BPA biodegradation occurred at the lowest concentrations, in other words those present in wastewaters given the small water solubility of this compound. The results are discussed in terms of the process of thermodialysis by considering the additional BPA fluxes towards the immobilized enzymes driven by the temperature gradients.

The behaviour of three different laccase-based graphite biosensors was studied in view of their use in agricultural or industrial waters polluted by phenolic compounds. Catechol was used as a substrate model. Laccase from Trametes versicolor was immobilized on one biosensor (type A electrode) by adsorption while, on the other two biosensor types, laccase was covalently bound through the carboxylic groups created on the graphite by means of treatment with an electric potential difference (type B electrode) or with nitric acid (type C electrode). In the latter two cases, hexamethylenediamine and glutaraldehyde were used as the spacer and the coupling agent, respectively. The extension of linear response range and the sensitivity and time stability of each biosensor type were investigated. The type C biosensor gave the best results and its electrochemical properties proved comparable to those reported by other authors.

A modified polyethersulphone graft membrane was loaded with antiproteases, with the aim of reducing the active protease blood concentration during hemodialysis in acute catabolic renal failure or cardiopulmonary bypass. As protease/antiprotease system, elastase and α 1 -antitrypsin were used. The concentration of active elastase in aqueous solutions decreased as function of contact time with the membrane, approaching saturation. A 40% loss of elastase activity was obtained at pH 7.4, which was not due to autolysis, which accounted for 5% of the loss. The highest reduction was achieved at pH 9.0 (25% higher than at pH 7.4). The saturation level of elastase decrease, calculated by means of the Einstein equation, was reached after more than 47 minutes. We speculate that a time reduction might be achieved either increasing the concentration of immobilized antiproteases, or increasing the rate of elastase movement across the membranes by hydraulic, osmotic, or temperature gradients. This technology can be applied to hemodialysis, and in extracorporeal blood circulation to promote elastase release.

A modified polyethersulphone graft membrane was loaded with antiproteases, with the aim of reducing the active protease blood concentration during hemodialysis in acute catabolic renal failure or cardiopulmonary bypass. As protease/antiprotease system, elastase and alpha1-antitrypsin were used. The concentration of active elastase in aqueous solutions decreased as function of contact time with the membrane, approaching saturation. A 40% loss of elastase activity was obtained at pH 7.4, which was not due to autolysis, which accounted for 5% of the loss. The highest reduction was achieved at pH 9.0 (25% higher than at pH 7.4). The saturation level of elastase decrease, calculated by means of the Einstein equation, was reached after more than 47 minutes. We speculate that a time reduction might be achieved either increasing the concentration of immobilized antiproteases, or increasing the rate of elastase movement across the membranes by hydraulic, osmotic, or temperature gradients. This technology can be applied to hemodialysis, and in extracorporeal blood circulation to promote elastase release.

Catalytic membranes, obtained by immobilizing thermophilic beta-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic beta-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, alpha and beta, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1 degrees C or a temperature gradient of 1 degrees C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed.

A planar bioreactor, equipped with a polypropylene membrane on which a lipase was immobilised, has been employed in a bioremediation process involving water polluted by dimethylphthalate (DMP), a model for a class of endocrine disruptors. The dependence ofenzymeactivity on pH, temperature andDMP concentration has been characterised under isothermal conditions, whereas the kinetics parameters have been studied under non-isothermal conditions. The following sequence was found for the values of lipase affinity, Km, towards the DMP: K(free-m) < K(imm-m,non-isoth) < K(imm-m,isoth). A comparison of the results obtained under isothermal and non-isothermal conditions indicated that there was an advantage in using non-isothermal bioreactors in the environmental field. These advantages in particular resulted in: (i) an increase in the enzyme activity proportional to the applied transmembrane temperature difference and (ii) a reduction in the bioremediation times and, consequently, the process costs. The advantages in using bioremediation processes in the place of classical membrane processes, such as ultrafiltration or reverse osmosis, are also discussed.