Brennan, Mark (1995) The whole cell catalysed hydrolysis of acrylamide to ammonium acrylate using an immobilised cell bioreactor. Doctoral thesis, University of Huddersfield.

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.

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