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CONTINUOUS ENZYMATIC LUBRICANT PRODUCTION FROM FUSEL OIL IN
A TWO-STAGE PACKED BED REACTOR INCORPORATING A WATER
EXTRACTION COLUMN
Renata N. Vilas Bôas¹, Rosemar Lima¹, Pedro C. Oliveira¹, Heizir F. de Castro¹
1 Escola de Engenharia de Lorena - USP, Departamento de Engenharia Química
E-mail: heizir@usp.br
ABSTRACT
The esterification of fusel oil with oleic acid catalyzed by lipase from Rhizopus oryzae immobilized on
polystyrene/divinylbenzene copolymer resin was assessed in a two-stage packed bed reactor running
on a continuous flow. Molecular sieves were used to remove the water formed as byproduct by
coupling the reactor with a column extractor. The modification presented herein introduces a
pathway for replacing saturated molecular sieves by regenerated sieves at a given time. Ester
synthesis on a continuous flow carried out without a water column extractor was used as a control.
The performance of the proposed system was quantitatively confirmed for space-times of 10 h, thus
resulting in average productivities of 292.2 ± 18.5
molester g-1 min-1, with minimal feedstock lost and
good stability of the immobilized lipase, revealing an average half-life time of 179.6 h.
1. INTRODUCTION
Fusel oil is a byproduct from the sugarcane-to-ethanol process, being composed primarily by isoamyl
alcohol (> 65 wt %). Despite its exploitation does not act directly on reducing the final cost of the
alcohol, its application as feedstock in manufacturing industries, surely, represents an important step
to increase the overall efficiency of the ethanol industries (Patil et al., 2002). Studies suggest several
alternatives to use fusel oil, from which its application as feedstocks in esterification reactions seems
to be the best choice on account of the variety of esters that can be obtained depending on the acyl
donor such as acetic acid, butyric acid, caprylic acid, lauric acid and oleic acid (Vilas Boas et al., 2017;
Vilas Boas et al., 2018).
Traditional esterification processes make use of strong acid catalysts at high temperatures to favor
ester production; unfortunately, these conditions often produce undesirable byproducts that must
be removed by energy-intensive purifications. In contrast, enzyme-catalyzed reactions have become
a valuable alternative to overcome those restraints, providing a catalyst that is able to operate under
mild reaction conditions with reduced formation of byproducts and beyond that meets the green
chemistry criteria (Manley et al., 2008). Among the enzymes applied in biotechnological processes,
lipases (triacylglycerol hydrolase, EC 3.1.1.3) are the most efficient biocatalysts used for ester
synthesis mostly because of its high catalytic activity in organic media (Choi et al., 2015).
The present study aimed to synthesize isoamyl oleate ester from low-cost raw material (fusel oil)
using lipase as a catalyst. In order to obtain high volumetric productivities a packed bed reactor (PBR)
running on continuous flow was selected. This system has low energy consumption and lower reactor
volume due to the high enzyme/ substrate ratio compared to a conventional batch reactor. However,
continuous esterification reactions usually show limit performance since the water formed as
byproduct should be eliminated to avoid the gradual decrease on the ester formation due to the
shifting towards hydrolysis reaction. Therefore, in the present study attention was paid to obtain
isoamyl oleate on a continuous flow using PBR in series with a water column extractor packed with
molecular sieves and its performance compared with a control system (absence of water removal).
2. MATERIAL AND METHODS
All assays were performed with Rhizopus oryzae lipase acquired as an enzyme powder (Lipase 36P,
Biocatalysts, Cardiff, UK) and subsequently immobilized on polystyrene/divinylbenzene copolymer
resin (Diaion HP-20 resin, Sigma-Aldrich), attained average hydrolytic activity of 2500 ± 120 U.g-1.
Oleic acid was purchased from Sigma Aldrich (98%) and fusel oil was obtained from an ethanol
distillery (São Paulo) with a composition of 65% isoamyl alcohol, 8% isobutyl alcohol, 0.4% of n-butyl
alcohol, 1.3% of n-propyl alcohol and 9.2% of ethyl alcohol. The esterification reactions were carried
out in a two-stage packed bed reactor (D = 15 mm, L = 55 mm and V = 10 mL) coupling with a water
column extractor using feed medium consisting of fusel oil and oleic acid at molar ratio of 1:1.5 in
the presence of iso-octane. Each column was packed with 2.60 g immobilized lipase and the column
between them contained 7.30 g molecular sieves that corresponded to a mass ratio immobilized
lipase: molecular sieves of 1:3 (Figure 1). The saturated molecular sieves were replaced by
regenerated ones at least every two space-times.
Figure 1. Experimental set up PBR in series
connecting with water column extractor: 1
bath for temperature control; 2 feeding
reservoir; 3 peristaltic pump; 4, 6 enzyme
packed columns; 5 column with molecular
sieves; 7 product reservoir; 8 (dotted line)
water current; 9 (continuous line) feeding
current.
The progress of esterification was monitored by collecting samples at the exit of both columns and
the consumption of isoamyl alcohol and formation of isoamyl oleate were determined by gas
chromatography (Vilas Bôas et al., 2018). The oleic acid consumption was determined by titration
with 0.05 mol L-1 KOH and phenolphthalein as indicator. The water content formed during the
reaction was determined by Karl Fischer (Koehler Model AKF5000).
3. RESULTS AND DISCUSSION
The PBR was run without using any strategy to remove the formed water and the results show that
the steady-state was reached within 12 h and the isoamyl oleate formation remained stable in 0.75
± 0.08 mol L-1 up to 24h. Then, a gradual decreased in isoamyl oleate formation was verified, attaining
an ester concentration lower than 0.34 mol L-1 at 48 h of reaction. After this point, there was an
increase of both starting materials concentrations and, thus, a decrease in ester synthesis. These
results are due to the accumulation of water within the PBR (water levels increased from 0.05 to
0.29% wt.) after 48 h run, which changed the medium polarity and shifted the chemical equilibrium
towards the ester hydrolysis.
By using the experimental outline and running the system at flow rate of 1.6 mL h -1 (space-time=
10.2 h), the steady state was also attained in 12 h, and the ester concentration remained at an
average 0.93 ± 0.06 mol L-1 up to 72 h operation, following by gradual decreasing after this point
(Figure 2). This experimental set up showed to be more stable allowing running the system under
steady-state conditions for 3 days, in contrast with one day observed with the one stage reactor
(control). It was possible to replace the saturated molecular sieves by regenerated one every two
space-times, so that the second column was fed with medium containing much lower water contents.
As can be verified in Figure 3, the water level in the outlet from the first column reactor was gradually
increased during the course of the reaction while in the second column the water level remained
almost constant to levels lower than 0.4% wt., which favored the ester synthesis.
Figure 2. Profile formation of the isoamyl oleate running
on continuous flow control reaction (first column,
second column).
Figure 3. Water contents in the two stage packed bed
reactor before (white) and after (grey) the water column
extractor.
This strategy also allowed to maintain the activity of the biocatalyst during the continuous
operation as summarized in Table 1 the half-life time (t1/2) and inactivation constants (kd) of the
immobilized biocatalyst estimated for each experimental set up (one stage and two-stages PBR). The
results showed that molecular sieves play an important role on the reaction progress acting as
receptor agent of the water formed as by-product. In addition, the polarity of the reaction medium
is an important factor for the partitioning of water between the liquid and solid phases, which can
be affected by the biocatalyst stability when insufficient amounts of molecular sieves were used.
Table 1. Operational stability of R. oryzae lipase immobilized on STY-DVB under continuous
esterification reactions carried out in PBR.
Experimental set up
Biocatalyst half-life
(h)
Deactivation constant
(h-1)
Control reaction
59.3
0.012
PBR in series with water column extractor
179.6
3.85 x 10-3
The product obtained was purified and the properties determined with respect to kinematic viscosity
at 40 °C (6.95 mm2.s-1), density (820.6 kg.m-3), viscosity index (171) and water content (0.06%) were
similar those described in the literature for isoamyl oleate (Dormo et al., 2004).
4. CONCLUSIONS
This work demonstrated the potential for production of biolubrificant by esterification reaction on
continuous flow. Under the conditions established, it was possible to attain productivity of 292.2 ±
18.5 µmol isoamyl oleate g-1 min-1 and satisfactory operational stability of the biocatalyst (t1/2 =179.6
h), demonstrating a good performance and high potential of the tested experimental lay out.
5. REFERENCES
Choi, J., Han, S., Kim, H. 2015, Industrial applications of enzyme biocatalysis: Current status and future aspects,
Biotechnol. Adv. 33, 1443-1454.
Dormo, N., Belafi-Bako, K., Bartha, L., Ehrenstein, U., Gubicza, L., 2004. Manufacture of an environmental-safe
biolubricant from fusel oil by enzymatic esterification in solvent-free system. Biochem. Eng. J. 21, 229–234.
Manley, J.B., Anastas, P.T., Cue Jr, B.W., 2008. Frontiers in green chemistry: meeting the grand challenges for
sustainability in R&D and manufacturing. J. Clean. Prod. 16, 743–750.
Patil, A.G., Koolwal, S.M., Butala, H.D., 2002. Fusel oil: composition, removal and potential utilization. Int. Sugar J.
104(1238), 51–58.
Vilas Bôas, R.N, Biaggio, FC, Giordani, D.S, De Castro, H.F., 2017. Enzymatic synthesis of isopentyl caprylate using fusel oil
as feedstock. Quim. Nova 40, 541–547.
Vilas Bôas, R.N, Ceron, A.A, Bento, H.B.S, De Castro H. F. 2018. Application of an immobilized Rhizopus oryzae lipase to
batch and continuous ester synthesis with a mixture of a lauric acid and fusel oil. Biomass Bioenergy 119, 61–68.
Acknowledgements: The authors are thankful to the support given by the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior - CAPES (Finance Code 001) and by the São Paulo Research Foundation - FAPESP (grant number
2016/10636-8)
