Biofuels in Perspective

136 Biofuels
(diglycerides) decreased with increasing water content and incubation time, indicating
that R. oryzae lipase promotes acyl migration of partial glycerides from the sn-2 to sn-1(3)
position. The free fatty acid content increased with increasing water content and incubation
time, while that of other components (MEs and TGs) was not affected.
8.4 Use of Intracellular Lipase as Whole-Cell Biocatalyst
Methanolysis reaction can be carried out using extracellular or intracellular lipases, but
extracellular lipases require purification by procedures that may be too complex for practical
use. Furthermore, enzymes recovered through such operations are generally unstable
and expensive. Consequently, there has been considerable research into the direct use of
intracellular enzyme as a whole-cell biocatalyst. 42–44 The present section describes the
whole-cell biocatalyst immobilization technique applied when utilizing R. oryzae intracellular
lipase, the effect of cell membrane composition on methanolysis reaction, and the
lipase localization within the cells.
8.4.1 Immobilization by BSP-Technology
To utilize the whole-cell biocatalyst in a convenient form, cells should be immobilized in
such a way that they resemble the ordinary solid-phase catalysts used conventionally in
synthetic chemical reactions. Of the many immobilization methods available, the technique
using porous biomass support particles (BSPs) developed by Atkinson et al. 45 has several
advantages over other methods in terms of industrial application: (1) no chemical additives
are required, (2) there is no need for preproduction of cells, (3) aseptic handling of particles
is unnecessary, (4) there are high rates of substrate mass transfer and production within
BSPs, (5) the particles are reusable, (6) the particles are durable against mechanical shear,
(7) bioreactor scale-up is easy, (8) costs are low compared to other methods. Because of its
advantageous features, the BSP technique has been applied successfully in a wide variety
of microbial, animal, and plant cell systems. 46
In Figure 8.5, the extracellular and intracellular (whole-cell biocatalyst) lipase production
processes are compared. 19 Unlike in the case of extracellular lipase, no purification or
immobilization processes are needed in preparing whole-cell biocatalysts with BSPs, since
immobilization can be achieved spontaneously during batch cultivation.
Utilizing R. oryzae cells immobilized within BSPs as whole-cell biocatalyst, Ban et al. 47
investigated the culture conditions for lipase production and the effects of cell pre-treatment
and the water content of the reaction mixture on methanolysis in a 50-ml screw-cap bottle
incubated on a reciprocal shaker. As shown in Figure 8.6, the R. oryzae cells were readily
immobilized within the polyurethane foam BSPs during batch cultivation. Addition of
olive oil or oleic acid to the culture medium as a substrate-related compound significantly
benefited the intracellular lipase activity, while no glucose was necessary. As a result, when
methanolysis was carried out with stepwise .addition of methanol using BSP-immobilized
cells in the presence of 10–20 % water, the ME content of the reaction mixture reached
80–90 % with no organic solvent pretreatment. 47 This level of ME production is almost the
same as that achieved using extracellular lipase. 22 Acyl migration can thus occur not only
when extracellular lipase is used but also with R. oryzae cells as the whole-cell biocatalysts.