The whole cell catalysed hydrolysis of acrylamide to ammonium acrylate using an immobilised cell bioreactor
Methods currently used for manufacturing the commodity chemical ammonium acrylate involve high temperatures that increases the risk of unwanted polymerisation and, in certain cases, leads to the generation of large amounts of unwanted by-product. The enzyme catalysed hydrolysis of acrylamide through to ammonium acrylate, however, may be carried out at ambient temperatures without by-product generation. Bioreactors operating with immobilised whole cell biocatalysts, have been examined as a means of producing ammonium acrylate. Studies with the amidase active C. nitrilophilus showed that entrapment in cross-linked polyacrylamide gel was the best immobilisation method, resulting in a biocatalyst with good physical stability without a serious loss in amidase activity. Immobilisation scaleup was possible through the use of a suspension polymerisation technique to produce cells entrapped in cross-linked polyacrylamide beads. The beads exhibited amidase activity after drying and rehydration. The loss in amidase activity was reduced by decreasing the drying time while storage stability was increased when the beads were dried to a low water content. Bioreactor studies were performed using C. nitrilophtlus entrapped in cross-linked polyacrylamide gel cuboids. The changing conductance of reaction solutions, due to ammonium acrylate production, was used as an on-line method of monitoring amidase activity. Interfacing the conductance monitor to the acrylamide feed system, via a computer, allowed a 0.5 litre continuous stirred tank bioreactor to be operated at constant acrylamide and ammonium acrylate concentrations for several days at a time. It was shown that batch reactors were unsuitable for ammonium acrylate production as amidase activity was progressively and irreversibly deactivated in the presence of acrylamide and, to a lesser extent, ammonium acrylate. Amidase activity was decreased at lower reactor operating temperatures whilst amidase stability was increased. The automated bioreactor system was used to compare the stability of the amidase activity of C. nitrilophilus with that of two cell isolates: R rhodochrous sp.632 and Rhodococcus sp.l068. The amidase activity of R rhodochrous sp.632 was shown to be the most stable. The amidase activity of R rhodochrous sp.632 was found to be competitively inhibited by ammonium acrylate. Use of a fed-batch reactor for ammonium acrylate production was preferred over a continuous stirred tank reactor as the effects of product inhibition were reduced. Through monitoring of the conductance measurements, the fed-batch system was automated so that acrylamide concentrations were kept at a constant level. Operation of the system at different acrylamide concentrations showed that higher concentrations increased the rate of amidase activity loss. Bioreactor scale-up was performed by designing, constructing and operating a stirred tank reactor system with a 6 litre working volume. The reactor was operated in fedbatch and continuous modes using computer control, and ammonium acrylate was produced on a kilogram scale. Performance of the 6 litre reactor operating with R. rhodochrous sp.632 immobilised in cross-linked polyacrylamide beads, was comparable to the performance of the 0.5 litre reactor. Performance tests on polymers prepared from the bio-ammonium acrylate showed them to be indistinguishable from polymers of chemical origin.