Multiphase Bioreactor Design

The potentials of systems where biocatalysts are suspended in mixtures of substrates and
water vapour are relatively unexplored in contrast to those where enzymes are placed
directly in aqueous or non aqueous solvents. One of the major results of initial studies on
such systems was to prove that biocatalysts, traditionally functioning in liquid systems,
were able to bind and to transform molecules present in a gaseous phase, since there was
only one example of an enzyme acting on gaseous substrates reported in the literature at
that time.
Hydrogenase is a unique enzyme whose substrate is gaseous hydrogen. Yagi and
collaborators (1969) have clearly demonstrated that hydrogenase in the dry state binds the
hydrogen molecule and renders it activated, resulting in parahydrogen-orthohydrogen
conversion, whereas aqueous protons do not participate in the reaction mechanism.
In a subsequent paper Kimura et al. (1979) have proven that, using purified
hydrogenase, it was possible to obtain not only the conversion and exchange reactions,
but also the reversible oxido-reduction of the electron carrier, cytochrome c3 with H2-
Nevertheless, some other examples of utilisation of the gas/solid system using either
entire cells or isolated enzymes can now be found in the literature (Kim and Rhee, 1992;
Uchiyama et al., 1992; Zilli et al., 1992; Yang and Russell, 1996) and were reviewed a
few years ago (Lamare and Legoy, 1993).
Some systems have already been developed for the use of enzymes where the natural
substrates are non gaseous, such as the horse liver dehydrogenase (Pulvin et al., 1986),
the Sulfolobus solfataricus dehydrogenase (Pulvin et al., 1988) the Pischia pastoris
alcohol oxidase (Barzana et al., 1987, 1989a, 1989b) and finally lipolytic enzymes
(Parvaresh et al., 1992; Robert et al., 1992; Lamare and Legoy, 1995, 1997, 1999;
Lamare, 1996).
Enzymatic solid/gas bioreactors described in the literature are mainly used for single-
step biotransformations, but some examples have been described which use entire cells
for multi-step biotransformations. Two main classes can thus be defined, the enzyme
solid/gas bioreactors and the microbial solid/gas bioreactors.

Enzyme solid/gas reactors

Ethanol oxidation in the gaseous phase has been studied in batch reactors using Pichia
pastoris alcohol oxidase (Barzana et al., 1987, 1989).
Dehydrated enzyme immobilised on DEAE cellulose or on controlled pore glass beads
has been shown to oxidise methanol and ethanol vapors at elevated temperatures, in the
absence of water in the gas phase. Nevertheless, the study on the effect of water activity
showed that enzyme activity in the gas phase increases by several orders of magnitude,
whereas the thermostability decreases drastically when aw is increased from 0.11 to 0.97.
These studies yielded two important results. Firstly, the enzyme is active on gaseous
substrates even at hydration levels below the full hydration of the protein. Secondly, there
exists an antagonistic effect concerning the water activity. The higher the water activity,
the higher the reaction rate, but the stability is lowered at elevated temperatures.
Considering the reaction scheme of alcohol oxidase:

Multiphase bioreactor design 262