| dc.description.abstract | The enzymatic production of biodiesel is a promising alternative to the conventional production
by the alkaline route in a homogeneous medium. The enzymatic process allows using raw
material with any concentration of free fatty acids due to the specificity of the lipases and lower
energy consumption, because the enzymes act at mild temperature conditions. The liquid
formulations of lipase from Thermomyces lanuginosus, Eversa® Transform and Eversa®
Transform 2.0, were launched by Novozymes A/S to be used in the biodiesel production in their
free forms. After characterizing the two formulations, it was identified that both are very
similar, differing in the greater thermal stability of the second formulation, which is used to
give segment to this work. The immobilization of enzymes can improve theior performance in
the production of biodiesel, in addition to enabling the recovery and reuse of the biocatalyst. In
this work, Eversa lipase was immobilized by the technique of cross-linked enzyme aggregates
(CLEAs), a simple, economical and unsupported technique, capable of generating insoluble
biocatalysts with high volumetric activity and improved stability. The preparation conditions
were optimized in order to improve the properties of the biocatalyst. The nature of the
precipitant and concentration of the crosslinking agent were evaluated. To improve the
crosslinking step, bovine serum albumin (BSA), soy protein (SP) or polyethyleneimine (PEI)
were co-precipitated and evaluated as co-feeders and additives. Starch (later enzymatically
degraded) was used as a porogenic agent to reduce the diffusion limitations of the substrate.
Magnetic silica nanoparticles functionalized with amino-octyl groups (MNPS) were also used
to simplify the handling of CLEA, which are incorporated into the CLEAs by inter and/or intra
crosslinks particle-enzyme with glutaraldehyde as bifunctional agent, but a great percentage of
enzyme could also to be adsorbed on the nanoparticle surface before the aggregation step. The
best CLEA was prepared using PEI, starch and MNPS (Eversa m-CLEA). Under these
conditions, the biocatalyst showed an immobilization yield of 98.9%, recovered activity of
30.1%, a porous structure, and a thermal stability at 70°C around 40-fold higher than that of the
free enzyme. In the transesterification reaction of refined soybean oil with anhydrous ethanol
(molar ratio oil/ethanol 1:6), the best performance obtained was using 12 Uest/oil of Eversa m-
CLEA at 40ºC. A mass yield of 89.8% of fatty acid ethyl esters (FAEE) was found after 12 h
of reaction, while the soluble enzyme required 48 h of reaction to give the same yield. A caustic
polishing step of the product yielded a biodiesel containing 98.9 wt.% of FAEEs and a content
of free fatty acids (FFAs) below 0.25 wt.%, meeting the international standards of a biodiesel
to be commercialized as biofuel. The immobilized biocatalyst could be reused for at least five
cycles of 12 h, maintaining 89.6% of the first-cycle mass yield, showing the efficient recovery
of the catalyst by applying an external magnetic field. From an experimental design (DCCR)23 ,
it was possible to define a range of optimized values to produce enzymatic biodiesel using also
Eversa m-CLEA, however in the transesterification of raw materials oil, degummed soy oil,
and hydrated ethanol. At a molar ratio oil/ethanol of 1:6, 4 Uest/oil at 40 ºC, a mass yield of
81.45 wt.% of FAEEs, with a FFAs content of 3.9 wt.% was obtained after 24h of reaction.
After a caustic polishing, a yield of 89.88 wt.% of FAEEs with a FFAs content of 0.17 wt.%
was obtained. The use of Eversa m-CLEA, a biocatalyst that is easy to recover and reuse,
together with unrefined raw materials with lower added value, is a promising combination to
contribute to the economic viability of the enzymatic route, aiming to make it competitive to
the traditional alkaline route. | |
