Accepted Manuscript

Title: Lipase catalysed biodiesel synthesis with integrated

glycerol separation in continuously operated microchips
connected in series

Authors: Anita Šalić, Ana Jurinjak Tušek, Aleksandra Sander,

Bruno Zelić

PII: S1871-6784(17)30274-1
DOI: https://doi.org/10.1016/j.nbt.2018.01.007
Reference: NBT 1051

To appear in:

Received date: 8-6-2017

Revised date: 30-11-2017
Accepted date: 28-1-2018

Please cite this article as: Šalić, Anita, Tušek, Ana Jurinjak, Sander, Aleksandra,
Zelić, Bruno, Lipase catalysed biodiesel synthesis with integrated glycerol separation
in continuously operated microchips connected in series.New Biotechnology
https://doi.org/10.1016/j.nbt.2018.01.007

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Lipase catalysed biodiesel synthesis with integrated glycerol separation in

continuously operated microchips connected in series

Anita Šalić1, Ana Jurinjak Tušek2, Aleksandra Sander1, Bruno Zelić1*

1
University of Zagreb, Faculty of Chemical Engineering and Technology, Marulićev trg 19,

HR-10000 Zagreb, Croatia

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2
University of Zagreb, Faculty of Food Technology and Biotechnology, Pierottijeva 6, HR-

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10000 Zagreb, Croatia

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(asalic@fkit.hr, atusek@pbf.hr , asander@fkit.hr, bzelic@fkit.hr)

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*
Corresponding author: U
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Bruno Zelić
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e-mail: bzelic@fkit.hr
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Tel: + 385 (0)1 4597 146

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Fax: + 385 (0)1 4597 133

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Highlights

 Integrated system for biodiesel synthesis with integrated glycerol separation was
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developed
 A conversion of 95% was obtained within residence time of 2h
 Deep eutectic solvent was used to extract the biodiesel
 Purified biodiesel was produced with glycerol content under detection limit
Abstract:

Although the application of microreactors in different processes has been extensively

explored in recent decades, microreactors continue to be underexplored in the context of the

enzyme-catalysed process for biodiesel production. Due to their numerous advantages,

microreactors could become the next step in the development of a biodiesel production

process characterised by sustainability, cost-effectiveness and energy efficiency. In this

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investigation, biodiesel production was catalysed by lipase from Thermomyces lanuginosus

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(Lipolase L100). Edible sunflower oil was used as a model substrate in order to investigate

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the process. After optimal process conditions had been determined, waste-cooking oil was

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used for biodiesel production to make the production process more sustainable. Three

different substrate-feeding strategies were investigated and finally an optimal strategy was
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proposed. In all the investigated systems, fatty acids methyl esters (FAME) content was
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higher than 95% and obtained in a significantly shorter time (less than 2 h) compared to the
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batch process in which biodiesel production was catalysed by lipase (C = 95%, t = 96 h).
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After the optimal biodiesel production system had been proposed, an integrated system with
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two microchips connected in series was developed. The first microchip was used for biodiesel

production and the second for simultaneous purification i.e. glycerol separation. Finally,
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purified biodiesel was produced with glycerol content below the detection limit.
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Keywords: biodiesel, microreactor, biocatalysis, lipase, DES, integrated system

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Introduction
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Nowadays, as the availability and accessibility of fossil fuels is significantly declining, the
need for the production of biofuels from various renewable sources is becoming increasingly
interesting. Compared to conventional fuels, biodiesel has the following main advantages:
increased biodegradability, increased lubricity, high flash point, non-toxicity, and reduced
emissions of hydrocarbons, sulphur, carbon monoxide and particulate matter. All these
advantages make biodiesel an environmentally friendly fuel [1-4]. There are a variety of
methods for biodiesel production, e.g. microemulsification [5], pyrolysis [6-10], and
transesterification [11-15]. Today’s industrial production of biodiesel is mostly carried out in
batch reactor systems applying the transesterification process (also known as alcoholysis) of
edible vegetable oil and animal fats (the first generation of biofuels), waste edible oil
remaining after frying and non-edible oil (the second generation of biofuels) [16] with short-
chain alcohols such as methanol and ethanol. The main disadvantages manifested by these
conventional methods of biodiesel production include long residence times of the reaction

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mixture, high energy consumption and low process efficiency [1, 17-19]. According to

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Budžaki et al. [20], the ideal biodiesel production process should include continuous

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production with stable catalysts and with minimization and/or elimination of product
separation and purification steps. Such ideal technology has not yet been developed. In order

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to address the above issues, and further justify ecologically and economically and enhance
the process of biodiesel production, improvements to the existing and the development of

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new technologies are being fostered intensively. The main features of the newly developed
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processes need to be an increase in the reaction rate, reduction of the molar ratio of alcohol to
oil in the reaction mixture, and low energy consumption, resulting from the efficient mass
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and heat transfer [21].
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Microreactor systems are one of the emerging technologies that could become suitable for
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overcoming the limitations of the currently used processes of biodiesel production and, in
addition, their reduced dimensions significantly lower the implementation and operating costs
[22]. Besides the cost reduction, a high surface to volume ratio, faster diffusion dominated
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transport, enhanced heat transfer and consequently reduced energy demands, good process
control, high throughput, and usage of minimal (microlitres) reagent volumes, etc., are some
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advantages of the microreactor that are usually highlighted and could be exploited in biodiesel
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production [23]. Furthermore, they are known to considerably increase the dispersion of two
phases needed for biodiesel reactants (alcohol and oil; [20]). Xie et al. [24] outlined the main
features of biodiesel production in microreactors compared to the conventional reactor
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systems: a 4,167-fold smaller volume of the reactor, 60% reduction in total plant size, 1,678-
fold higher surface area to volume ratio, 4,167-fold higher productivity, 18 times less energy
consumption, 4,554-fold higher mass and heat transfer, 70,000 fold higher efficiency of
mixing, 24.4% savings in capital costs and 11.1% savings in production costs. Moreover,
Budžaki et al. [20] recently made an analysis of biodiesel production in the face of industrial
production, traditional methods, novel technologies and recent progress in biodiesel
production. They commented critically on the suitability of microreactors for the biodiesel
production laboratory and industrial scale, weighing up all the advantages and disadvantages
and concluding that there might be a future for a biodiesel/microreactor/enzyme combination
process.

To date attempts have been made to apply the microreactor technology in biodiesel
production, but they were all mostly limited to the use of chemical catalysts [24]. However,

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mild reaction conditions, absence of unwanted by-products (soap), reusability, simple

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separation and purification of the resulting biodiesel as well as lower energy consumption are
some of the many features that make the enzyme a better choice than traditional chemical

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catalysts in the biodiesel production process [25-27]. Lipases are among the most versatile

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catalysts for biodiesel synthesis. They were originally defined as a special group of esterases
which exhibit the phenomenon of so-called interfacial activation, deviating from classical

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Michaelis-Menten kinetics. They are activated at the water-lipid interface and their very low
activity on monomeric substrates increases strongly when the substrate reaches concentrations
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over its supersaturation level, where lipids may exist as an emulsion or in the form of a
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micelle [28]. Due to this property, lipase-catalyzed transesterification of oil feedstocks has
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been considered one of the most promising enzymatic techniques for producing biodiesel and
their application has been reported in many publications, e.g. [29-36]. They are used in
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biodiesel production due to their ability to simultaneously catalyse hydrolysis, esterification

and transesterification [27]. Although lipases from yeast of genus Candida are the most
popular, some authors [37] claim that lipases from Thermomyces lanuginosus (TlL) are very
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useful enzymes in many processes. TlL lipases are basophilic and noticeably thermostable,
and commercially available in both soluble and immobilized forms. Initially, they were
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oriented toward the food industry, but have found applications in many different industrial
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areas, from biodiesel production to fine chemicals, having even better performance in some
instances than lipases from other sources. One of those processes is biodiesel production in
which the high stability of TIL has a significant effect [37]. TlL also happens to be among the
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least expensive commercially available lipases [38].

As mentioned above, TlL is available in both soluble and immobilized forms and both can be
used for biodiesel production. Both forms have their advantages and disadvantages. Proper
immobilization may greatly improve the enzyme features, from stability to activity and
resistance to stirring, as well as decreasing inhibitions and the effect of chemical
modifications, etc. [39]. Moreover, enzyme reuse is simpler when immobilized [40-41]. On
the other hand, diffusion is one of the main problems of immobilized enzymes [42, 43].
Furthermore, in biodiesel production, the accumulation of reaction by-products (water and
glycerol) in the support material is the main reason for lipase inactivation during operation, in
addition to the negative effect of alcohols (mainly methanol) on enzyme stability [44-46]. The
last two drawbacks encouraged the use of free enzymes as an alternative to solve the problems
generated by the support [47-49].

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Until now, TlL has been tested in biodiesel production [50-56] on a macro scale, but despite

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numerous advantages such as the enormous potential for reducing energy requirements and
environmental issues, enzymatic methods are still not able to compete with chemical

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processes. The main obstacle for their introduction is the high cost of enzyme and its reduced

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activity and stability in the presence of a polar alcohol such as methanol and ethanol. Some of
these deficiencies could be resolved by applying microreactors, which are still underexplored

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in the context of the enzyme-catalysed process of biodiesel production.
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Once biodiesel is produced, and could be used in internal combustion engines, it has to fulfil
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the quality parameters prescribed by the American standard ASTM D 6751 [57] and the
European standard EN 14214 [58]. The purity of biodiesel is obtained through the purification
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process that involves the removal of excess alcohol (methanol), unreacted triglycerides,
glycerol and catalyst and the resulting soap, the share of which in biodiesel depends on the
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raw material from which biodiesel is produced and on the catalyst used. The conventional
purification methods include water washing of biodiesel (also known as wet washing), dry
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washing and membrane separation. However, water rinsing generates large quantities of
wastewater that must be properly disposed and represents a great economic and
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environmental burden [59]. In addition, it results in a loss of biodiesel during the washing
phase. To overcome these limitations, alternative methods of purification without using water
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(dry washing) have been developed. The methods include the use of different materials such
as absorbents, solvents and ion exchangers based on a variety of resins [60]. Recently, of
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those “alternative” methods, deep eutectic solvents (DES) have gained a lot of attention
among researchers because they resemble various unique characteristics such as low
volatility, non-flammability and non-toxicity. Another major advantage is that a large
diversity of them can be prepared easily using affordable raw materials [61]. In the context of
biodiesel purification, it has been shown that a DES synthesized using choline chloride salt
and ethylene glycol was able efficiently and fully to remove free glycerol from biodiesel [60,
62-64].

In this study, the use of tubular microreactors for biocatalytic biodiesel production was
explored. Biodiesel production was performed by transesterification of edible and waste
cooking oil using the commercial lipase as a biocatalyst. The free enzyme was used to avoid
some of the disadvantages of the immobilized enzyme – diffusion and accumulation of
reaction by-products when the support is used and low immobilization efficiency when the

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enzyme is immobilized on a channel surface [65]. Since microreactors are continuous flow

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reactors, most of the proposed and tested supports would be washed out of the reactor. A hint
for future investigation would be the implementation of magnetic particles as the support for

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enzyme immobilization that could be entrapped in a reactor by a simple magnet [66]. Three

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different substrate feeding strategies were investigated: a) oil and enzyme dissolved in a
buffer in the first phase and methanol in the second phase, b) methanol and oil in the first

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phase and the enzyme dissolved in a buffer in the second phase, and c) methanol and enzyme
dissolved in a buffer and oil in the second phase, and the optimal strategy was proposed for
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biodiesel production in a microreactor. Since oil and buffer (aqueous phase), or oil and
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alcohol, do not make stable solutions which lead to the phase separation in the syringe and
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unequal substrate distribution in a microchannel, several emulsifiers were tested. As some of

the chosen emulsifiers belonged to the detergents group, and knowing that detergents can
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have a negative effect on TlL stability and activity (due to their close resemblance to lipase
substrates) [67-69] when choosing optimal emulsifiers/detergents, the enzyme activity was
tested in their presence.
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As a final step, an integrated system composed of biodiesel production with simultaneous

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purification using choline chloride ethylene glycol as a purification medium was developed.
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Material and methods

Materials
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Chemicals

Edible sunflower oil (Zvijezda, Croatia), mustard (Podravka, Croatia), potatoes and eggs were
purchased from a nearby supermarket. Waste cooking oil (WCO) used in this research was
collected after deep-frying of potatoes. The lipase from Thermomyces lanuginosus (Lipolase
100L), heptane, F.A.M.E. mix GLC-10, sodium dodecyl sulfate (SDS), tween 80, triton X-
100, gum arabic, ethylene glycol, chloroform, dimethyl sulfoxide (DMSO), isoamyl alcohol
and 4-nitrophenyl acetate were purchased from Sigma-Aldrich Handels GmbH (Austria).
Methanol, tris(hydroxymethyl)aminomethane (TRIS), isopropanol, HCl, acetone and ethanol
were purchased from Kemika (Croatia). Acetonitrile was purchased from Fisher Chemicals
(United Kingdom). Polyethylene glycol 1550 (PEG 1550) and 6000 (PEG 6000) were
purchased from Merck (Darmstadt, Germany). Choline chloride was purchased from Acros
Organics (Belgium).

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Methods
Lipase assay

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The lipase activity was determined by the procedure described by Sörensen et al. [70] with the
exception of buffer composition. In all experiments performed here, Tris-HCl (0.05 mol/l)
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buffer pH 8 was used as in the procedure described by Palacios et al. [71]. Briefly, 1.485 ml
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of lipase solution (0.01 mg/ml) diluted in Tris-HCl buffer was added to a UV-cuvette. 50 μl of
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4-nitrophenyl acetate dissolved in acetonitrile was added to the cuvette and rapidly shaken.
The activity was determined by measuring the change of absorbance at 410 nm with a
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spectrophotometer (Shimadzu UV – 1601, Kyoto, Japan) in the interval of 60 s. All the

measurements were performed in triplicate and the results showed no significant difference in
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the 95% confidence interval.

Effect of emulsifier
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In order to prepare stable water:oil (W:O) and methanol:oil (M:O) emulsions, different
emulsifiers were investigated. The initial concentration of all selected emulsifiers was 1 g/l.
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The following were investigated: SDS, PEG 1550, PEG 6000, Tween 80, Triton X-100, gum
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arabic, mustard and egg white. The solutions were prepared by mixing oil either with water or
with methanol in 8:1 ratio after adding the selected emulsifier. The mixture was mixed in the
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laboratory shaker (Tehtnica, Vibrimix 313EVT, Czech Republic) for 5 m at 7,000 rpm and
then placed on a holder to observe the (in)stability of phases. The height measuring
instrument was placed next to the testing tubes and the phase separation speed was calculated
as a ratio of constant height (2 cm) and the time needed for the volume of the lower phase to
obtain the height specified.
Effect of organic solvents on the enzymatic reaction

In order to stop the enzymatic reaction at the outflow of the microreactor, different solvents
were investigated: methanol, ethylene glycol, isopropanol, acetonitrile, acetone, DMSO,
Marmur solution (chloroform:isoamyl alcohol = 24:1). The sample was mixed with the
organic solvent in a 1:9 ratio, rapidly shaken and the enzyme activity was measured according
to the procedure indicated above.

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In addition, according to one of the experimental plans the enzyme was intended to be in
contact with methanol for a longer period of time, and thus long-term exposure to methanol

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was also investigated. The enzyme dissolved in a buffer was mixed with methanol in a 1:9

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ratio. The samples were taken at different time intervals and the enzyme activity was
measured as described previously.

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Deep Eutectic Solvent (DES) preparation
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Choline chloride and ethylene glycol were dried in a vacuum dryer at 60 °C for 24 h before
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use. DES, choline chloride ethylene glycol (ChCl:Etgl) was prepared by heating cholinium
salt and a hydrogen bond donor, in the ratio of 1:2.5 (molar ratio) in a rotary vacuum
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evaporator (250 mbar and 60 °C) until a homogeneous liquid had formed (T = 25°C,  =

1.1224 g/ml, η = 0.0356 Pas).

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Measurement of fatty acid methyl esters (FAME) and glycerol

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The FAME concentration was determined by the method of Budžaki et al. [27]. The samples
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were analysed by gas chromatograph Shimadzu GC-2014 (Kyoto, Japan) equipped with FID
and Zebron ZB-wax GC capillary column (length 30 m, I.D. 0.53 mm and film thickness 1.00
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μm). The method consisted of maintaining the temperature at 180 °C for 1 m and then
heating up to 230 °C at a rate of 5 °C/m. The total determination time was 20 m with helium
as a carrier gas at 1.97 ml/m. Peaks identification was carried out using the standard F.A.M.E.
mix GLC-10. The retention times for the corresponding esters of fatty acids were 7.494 m for
palmitic, 10.192 m for stearic, 10.545 m for oleic, 11.257 m for linoleic and 12.336 m for
linolenic acids. The same method was used for glycerol determination and the retention time
was 8.256 m.

Biodiesel synthesis in a microreactor

Biodiesel production from edible sunflower oil and waste cooking oil (WCO) using Lipolase
100L was performed in a polytetrafluoroethylene (PTFE, teflon) tubular capillary
microreactor (length:width = 500 mm:1 mm, internal volume 392.5 μl). Three different

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feeding strategies were investigated in order to determine the optimal approach to biodiesel

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production (Figure 1). All experiments were performed at 40 °C (optimal enzyme activity

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temperature) which was obtained by emerging the microreactor in a water bath with a heat
regulation system (Thermomix 1420, Braun, Germany). In the first experimental set-up, oil

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was placed into one syringe and fed from one inlet into the microreactor while methanol and
enzyme were mixed together and fed from another inlet using two syringe pumps (PHD 4400

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Syringe Pump Series, Harvard Apparatus, USA) equipped with high-pressure stainless steel
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syringes (8 ml, Harvard Apparatus, USA). In the second experimental set-up, oil and
methanol were mixed together with emulsifier and placed into one syringe while enzyme was
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placed into another. In the third experimental set-up, oil and enzyme dissolved in a buffer
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formed a stable emulsion after addition of emulsifier and were placed into one syringe, while
the methanol phase was put into the second syringe. The total flow (Φ = 1.25 – 400 μl/m) was
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altered and the influence on FAME formation was monitored. The outflows from
microreactors containing oil, methanol, FAME, water and enzyme were collected in vials
placed on ice in combination with organic solvent to stop the reaction via enzyme
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inactivation.
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Figure 1 here
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Biodiesel synthesis with integrated glycerol separation in continuously operated microchips

connected in series
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After selecting the optimal system for biodiesel production, an integrated system was
developed (Figure 2). It consisted of two microchips connected in series. The first microchip
– microreactor – was used for biodiesel production while the second one – microseparator –
was used for simultaneous glycerol separation, i.e. biodiesel purification. Methanol was fed
into the microreactor from one inlet while the mixture of SDS, enzyme and oil was fed from
the second inlet using two syringe pumps (PHD 4400 Syringe Pump Series, Harvard
Apparatus, USA) equipped with high-pressure stainless steel syringes (8 ml, Harvard
Apparatus, USA). The outlet of the microreactor was directed into the microseparator where
glycerol separation took place. ChCl:Etgl was used as a medium for glycerol removal and
was fed as the second inlet stream into the microseparator using the third syringe pump (PHD
33 Syringe Pump Series, Harvard Apparatus, USA). Both chips were heated to 40 °C in a
water bath (Thermomix 1420, Braun, Germany).

Figure 2 here

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The measurements in all experiments were performed in triplicate and the results showed no

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significant difference in the 95% confidence interval.

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Results and Discussion

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Stable emulsion formation

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In order to produce biodiesel in a microreactor, three different compounds have to be fed into
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the microchannel: oil, methanol and enzyme dissolved in a buffer (aqueous phase). As the
microreactor configuration consisted of two inlets, there are three different approaches that
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can be applied: to mix oil and enzyme dissolved in a buffer in the first phase and methanol in
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the second phase, to mix methanol and oil in the first phase and enzyme dissolved in a buffer
in the second phase, and to mix methanol and enzyme dissolved in a buffer in the first phase
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and oil in the second phase.

Since oil and buffer (aqueous phase) as well as oil and alcohol do not make a stable solution,
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prior to all experiments it was necessary to find an appropriate emulsifier to enable

preparation of a stable emulsion of two immiscible compounds. The formation of the stable
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emulsion was necessary because in preliminary experiments (data not shown), in which
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emulsifier was not used, phase separation in the syringe was observed resulting in unequal
distribution of phases (components) in the microreactor. By the use of emulsifiers such
problem could be avoided. As mentioned in the Introduction, some emulsifiers belonging to
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the detergents group can have an effect on the TlL activity and thermal stability. According to
references [67-69], activation at low detergent concentrations with inhibition at higher
detergent concentrations is present. The influence on activity was expected because of the
detergents resemblance to lipase substrates, both in terms of structure and complex
aggregation behaviour. The emulsifiers that were chosen were SDS, PEG 1550, PEG 6000,
Tween 80, Triton X-100, gum arabic, mustard and egg white and they were all tested in 0.1
g/l concentration. The results are indicated in Figure 3.

Figure 3 here

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Emulsion stability was assessed by visual observation as the phase separation speed. As can

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be seen from the results, only the emulsion of water, oil and SDS remained stable and
homogenous over a longer period of time (observation over a period of 7 d) (Figure 3a).

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Moreover, the emulsion produced by other emulsifiers exhibited phase separation with a clear

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layer at the top for water:oil system and on the bottom for alcohol:oil system in just a few
minutes. Based on the results, SDS for water:oil emulsion and Triton X-100 (Figure 3b) for
alcohol:oil emulsion were chosen. The influence of lower concentrations of SDS on the
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formation of a stable emulsion was also investigated. Concentrations of 0.125, 0.25 and 0.5
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g/l of SDS were investigated and in all phase separation was observed after 1 h. In the case of
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Triton X-100, higher concentrations were investigated to obtain a stable emulsion and for all
the explored concentrations (2, 3, 4, 10 and 20 g/l) no significant improvement of the stability
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was noticed.
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According to Palacios et al. [71] and Morgensen et al. [67], the choice of emulsifier can affect
the enzyme activity in that they can potentially cause denaturation of the enzyme thus
decreasing the activity, or they can increase activity by preventing enzyme agglomeration or
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have some effect on the closed-open equilibrium of individual lipase molecules. Furthermore,
some authors [68] claim that SDS was found to enhance the activity of TlL toward some
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substrates at premicellar concentrations of SDS, peaking at around the critical micelle

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concentration while above such concentration the activity declined but was still significantly
higher than in buffer alone, indicating that this aggressive detergent did not denature the
lipase. Therefore, the influence of Triton X-100 and SDS (both 1 g/l) on the enzyme activity
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was also investigated.

It was found that both emulsifiers had a negative effect on enzyme activity, implying that the
activity was decreasing in comparison to the experiment performed in buffer only without
addition of emulsifier (S.A.buffer = 760.93 ± 41.82 U/mg). The results for SDS were the same
as those obtained presented by Palacios et al. [71] in which a 20.60 % ± 1.43 reduction in the
activity was noticed. Nevertheless, the same authors reported complete enzyme deactivation
when Triton X-100 was used. In the present study a decrease of 53.91% ± 2.44 was found (the
enzyme was active), which is not in agreement with the results obtained in [71]. The results
obtained here were in correlation with other studies because Triton X-100 is commonly used
in lipase assays [72], which would not be possible if complete deactivation occurred.

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The effect of organic solvents on enzyme stability

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A completely opposite effect from the previous one was needed at the outflow of the

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microreactor in order to stop the enzymatic reaction. To achieve this, different solvents were

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investigated. All solvents were investigated in the ratio of solvent:sample = 9:1. The first was
Marmur solution [73], a combination of chloroform and isoamyl alcohol in a 24:1 ratio. This
is a common reagent applied in many biological studies of enzyme deactivation and proposed

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by Palacios et al. as well [71]. The results obtained are shown in Figure 4a.
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Figure 4 here
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The samples were analysed immediately after the organic solvent and the sample were mixed.
As can be seen, the reduction of activity to a mere 44.92%±1.02 was observed for Marmur
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solution, which was insufficient for instant enzyme deactivation. According to the dynamic
measurement (Figure 4b), it took about 36 m for activity to fall below 5% of the initial value
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(with the estimated deactivation constant of kd = 0.0640 ± 0.0003 m-1). Due to unsatisfactory
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results of the Marmur solution experiments, several different compounds with potential
enzyme deactivation ability found in references were investigated: methanol, ethylene glycol,
isopropanol, acetonitrile, acetone and DMSO [74, 75]. According to the literature data, lipase
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activity is usually compromised at organic concentrations above 60%. On the other hand,
according to the results in Figure 4a, the activity of lipase from Thermomyces lanuginosus
increased in the presence of ethylene glycol, isopropanol, acetonitrile and acetone, even if the
concentration of organic solvent was higher than 90%, while no effect (positive or negative)
was found in the experiments performed with methanol. Enzyme deactivation was noticed
only with addition of DMSO, but the effect was lower in comparison with that obtained with
Marmur solution. According to [75], it is poorly understood why the enzyme activity changes
in the mixtures of water and solvents. The authors state that numerous factors have been
suggested to bring such effects about, the commonest being: (i) water or organic solvents
might be one of the substrates; (ii) organic solvent molecules might act as specific inhibitors;
(iii) the organic solvents change the bulk properties of solvents such as dielectric constant,
polarity and hydrophobicity, etc., which in turn affects the solvation of substrate and/or
transition states and influences their binding to enzyme molecule; and (iv) organic solvents

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change the structure of the enzyme.

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As mentioned above, the denaturing effect of Marmur solution was not sufficient and thus, as

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a next step, the combination of thermal denaturation and addition of Marmur solution was

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tested. The results are given in Figure 4c. After the treatment with the mixture of ice and
Marmur solution, activity dropped below 5% (4.52%±1.91) of its initial value and was still

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decreasing (data not shown). Therefore, in all further experiments this mixture was used to
stop the enzymatic reaction at the outflow of the microreactor.
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A
Before performing the reaction in a microreactor, the influence of methanol on the enzyme
stability was tested over a longer period of exposure. This was performed using the first
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experimental set-up (Figure 1) in which methanol was mixed with enzyme dissolved in buffer
and placed into the syringe before introducing the mixture into the microreactor. In this way
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methanol and enzyme remained in contact for a long period of time. It was expected that
water-miscible solvents (like methanol) have a high tendency to strip off tightly bound water
PT

from the enzyme and the ability to partition deeper into the enzyme active site which in turn
causes a loss of both structure and activity [75]. The results are shown in Figure 5.
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Figure 5 here
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Although the results indicated that methanol does not have a negative effect on the enzyme
activity immediately after being exposed to it (Figure 4a), over a longer period of time the
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negative influence was observed (Figure 5). It was detected that after 3.5 h the activity
decreases by about 50% and after 15 h the enzyme becomes completely deactivated (kd =
0.0037 ± 0.00002 m-1).
Biodiesel production in a microreactor

As mentioned above, in order to investigate biodiesel production in a PTFE tube microreactor

with two inflows, three different feeding strategies were investigated (Figure 1).

In the first system, sunflower oil was fed into the microreactor as a separate phase, while
methanol and enzyme dissolved in a buffer were fed as the second inflow. All the experiments
were performed at the temperature of 40°C based on the results of Budžaki et al. [27]. The
results are indicated in Figure 6. The maximum FAME content (94.52% ± 3.38) was obtained

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for the residence time of less than 1 h. In comparison, the experiments performed in a stirred

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batch reactor [27] resulted in the same FAME content after 96 h. As suggested by Yeh et al.

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[76], although the simplest method to produce biodiesel is using a stirred batch reactor, the
duration of the entire reaction in such reactor generally requires several hours. The reason

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behind it is the immiscibility of oil and methanol that limits the reaction rate of the biodiesel
production process. Due to the small microchannel size (which assures a high surface to

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volume ratio, faster diffusion dominated transport, enhanced heat transfer and thus reduced
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energy demands, good process control, high throughput, etc. [23]), these limitations were
overcome resulting in faster performance. In addition, the amount of water in the reaction
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mixture can also have an impact on the reaction rate and FAME content. While water is
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essential in order to maintain the specific 3D structure of the enzyme [77], it is believed that
the excess of water floods the pores of the enzyme structure, which then decreases the enzyme
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exposure to the reaction media [78]. Excessive water can also enhance hydrolysis. In the
study by Budžaki et al. [27], the water concentration was around 10%, while in this study it
was below 5%. On the other hand, the proposed system had one major disadvantage.
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According to the results in Figure 5, when the enzyme was exposed to high concentrations of
methanol for a long period of time, it deactivated, for which reason this process is not suitable
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for continuous operation.

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A

Figure 6

In order to overcome this problem, it was necessary to separate methanol and enzyme at the
inlet. Therefore, two other. experimental set-ups were proposed: the second system, in which
methanol and oil were mixed together with Triton X-100 and the third one, in which oil was
mixed with a buffer (enzyme) containing SDS (based on the results suggested in Figure 3a) to
form a stable emulsion. The results are indicated in Figure 6. In all three experiments,
maximal FAME content above 94% was obtained for the residence time of approximately τ =
2 h. When all systems were compared, system I (experimental set-up I) was rejected because
of the negative methanol influence, while system II (experimental set-up II) was not stable
enough – the emulsion disintegrated too quickly (Figure 3b) and continuous mixing of
solution was necessary to obtain a homogenous mixture. Therefore, for the next step in this

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study, experimental set-up III was chosen. A possible disadvantage of this system is enzyme

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hydrolysis of oils that necessarily occured in syringe A during this experiment. Those

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reactions usually take longer periods, about 24 h for 50% of hydrolysis with stirring to
enhance mass transfer [79]. As a result, since the duration of experiments was shorter, no

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positive or negative effects were observed.

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Heating of the system (microreactor) is energy consuming and in order to reduce costs, the
experiment was carried out at room temperature (T = 21 °C) with the results outlined in
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Figure 6. In comparison to all other systems, the results were unsatisfactory because only 10%
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of FAME content was obtained. The prolonged residence time did increase the content, but in
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comparison to all other systems, did not occur rapidly enough. The reason behind this was
probably the negative impact of temperature on enzyme activity, especially taking into
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consideration that the optimal temperature for most lipases ranges from 30-60 °C [72].
Another reason could be the impact of temperature on oil viscosity and density which affected
the mass transfer.
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Another approach to make the process more cost-effective and environmentally friendly is to
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use waste cooking oil (WCO) available in significant amounts (1.85-2.65 million litres/d in
the EU [80,81]). WCO was filtrated before the reaction took place in the microreactor in order
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to remove all large food material and particles to avoid microchannel clogging. The reaction
was performed under the same conditions as described under experimental set-up III. WCO
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and the buffer (enzyme) were mixed together with SDS and fed into the microreactor as one
stream while the second stream consisted of methanol only. The reaction was performed at 40
°C and the results are indicated in Figure 6. Once again a high content of FAME (97.81% ±
2.18) was obtained. The residence time needed to obtain such content was surprising. Only 30
m was sufficient to obtain the same FAME content, which was significantly faster compared
to the reaction performed with edible sunflower oil, which took 2 h. The only difference
between those experiments was the source of oil. Consequently, the analysis of WCO and
cooking oils was performed and the amount of fatty acids compared. It was found that the
ratio between fatty acids changed after frying. While the amount of linoleic acid decreased,
the amount of other fatty acids, especially palmitic acid, increased (data not shown). In
comparison to the batch experiment [27], in which the maximum FAME content was obtained
after 96 h (82.57%), it can be concluded that microreactors should be considered as a good
and promising reactor system for enzymatic biodiesel production.

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The integrated system

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Once biodiesel is produced, it needs to be purified. To date, several separation and biodiesel

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purification devices have been developed. Liquid-liquid microseparators have been designed
to provide complete separation of organic and aqueous phases [82,83]. Most are based on wet
biodiesel washing [24, 84], which has many disadvantages already suggested in the

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Introduction. Here, an integrated system composed of biodiesel production with simultaneous
N
purification using choline chloride ethylene glycol as a purification medium was investigated.
A
As mentioned earlier, the first chip was used for biodiesel production and the second one for
simultaneous purification (Figure 2). A stable emulsion of enzyme dissolved in a buffer, oil
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and SDS was fed into the microreactor at a flow rate of Ф = 2.5 μl/m. Using the second
syringe pump, methanol was fed into the microchannel at the same flow rate. As the
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microreactor consisted of two inlets and one outlet, the outlet was directed into a second
microchip, in which the separation took place. Choline chloride ethylene glycol was used as
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the medium for biodiesel purification and was fed into the second microchip at a flow rate of
Ф = 5 μl/min. After purification, the sample was collected in the vials in which two clearly
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separate layers formed, the upper biodiesel layer and the lower DES layer. Biodiesel was
easily collected and analysed and DES was stored for further reuse.
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The biodiesel samples were analysed on GC according to the previously described method
A

and results, before and after purification, and compared. After the analysis of all samples, the
absence of glycerol was observed (glycerol concentration was below the detection limit for
the analytical method used). It was therefore concluded that the glycerol content in biodiesel
was below 0.02%, the maximum limit according to the American standard ASTM D 6751
[57] and the European standard EN 14214 [58]. The same procedure was repeated for
biodiesel produced from WCO with the same purification results. Accordingly, it was
concluded that the study resulted in efficient and successful integrated biodiesel production
followed by purification.

Conclusion

In this study, successful lipase-catalysed biodiesel synthesis with integrated glycerol

separation using DES in microchips connected in series was performed. As far as we are

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aware, this is the first time that the production and purification of biodiesel have been
performed in an integrated micro system comprising two microchips connected in series.

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Although the system is promising, there are some drawbacks that should be addressed in the

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future. One is certainly the enzyme reusability. At this point, the major issue is the price of

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enzyme and biodiesel, which is incomparable. There are several approaches to solve this
issue. One is the development of an integrated system with enzyme recirculation. The second
approach could be enzyme immobilization. The latter approach could solve not only the

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problem of enzyme reusability but also some other drawbacks of the system such as enzyme
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stability, which could be greatly improved. Consequently, one phase could be eliminated,
A
emulsifiers could be avoided, and so on. Additionally, owing to the mild reaction conditions
of the lipase catalysed reaction, glycerol as a by-product should be of pharmaceutical grade
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quality, and could be separated as a second product.

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CONFLICT OF INTEREST
The authors declare that there is no conflict of interest regarding the publication of this paper.
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Funding sources
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This work has been fully supported by Croatian Science Foundation under the project IP-
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2016-06-7993.
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Literature

[1] Demirbas A. Progress and recent trends in biodiesel fuels, Energy Convers Manage,
2009;50:14–34.
[2] Murayama T. Evaluating vegetable oils as diesel fuel, Inform, 1994;4:1138–45.
[3] Dunn RO, Shockley MW, Bagby MO. Improving low temperature flow performance of
biodiesel fuels and blends, Inform, 1994;5:529–32.
[4] Krawcyk T. Biodiesel – alternative fuel makes inroads but hurdles remain, Inform,
1996;7:801-15.
[5] Ma F, Hanna MA. Biodiesel production: A review, Bioresour Technol, 1999;70:1–15.
[6] Ates F, Pütün AE, Pütün E. Pyrolysis of two different biomass samples in a fixed-bed
reactor combined with two different catalysts, Fuel, 2006;85:1851–59.
[7] Zhang H, Xiao R, Huang H, Xiao G. Comparison of non-catalytic and catalytic fast

T
pyrolysis of corncob in a fluidized bed reactor, Bioresour Technol, 2009;100:1428–34.

IP
[8] Özbay N, Apaydin-Varol E, Uzun BB, Pütün AE. Characterization of biooil obtained from

R
fruit pulp pyrolysis, Energy, 2008;33:1233–40.
[9] Boateng AA, Mullen CA, Goldberg N, Hicks KB, Jung HG, Lamb JFS. Production of bio-

SC
oil form alfalfa stems by fluidized bed fast pyrolysis, Ind Eng Chem Res 2008;47:4115–22.
[10] Suurs AAR, Hekkert MP. Competition between first and second generation technologies:

U
lesson from the formation of a biofuels innovation system in the Netherlands, Energy,
N
2009;37:669–79.
[11] Kansedo J, Lee KT Bhatia S. Biodiesel production from palm oil via heterogeneous
A
transesterification, Biomass Bioenergy, 2009a;33:271–6.
M

[12] Kansedo J, Lee KT Bhatia S. Cerbera odollam (sea mango) oil as a promising non-edible
feedstock for biodiesel production, Fuel, 2009b;88:1148–50.
ED

[13] Jacobson K, Copinath R, Meher LC, Dalai AK. Solid acid catalyzed biodiesel production
from waste cooking oil, Appl Catal B – Environ, 2008;85:86–91.
PT

[14] Nakatani N, Takamori H, Takeda K, Sakugawa H. Transesterification of soybean oil

using combusted oyster shell waste as a catalyst, Bioresour Technol, 2009;100:1510–13.
[15] Guy MM, Lee KT, Bhatia S. Feasibility of edible oil vs non-edible oil vs waste edible oil
E

as biodiesel feedstock, Energy, 2008;33:1646–53.

CC

[16] Dragone G, Fernandes B, Vicente AA, Teixeira JA. Third generation biofuels from
microalgae. In: Current research, technology and education topics in applied microbiology
A

and microbial biotechnology, (Mendez-Vilas A, ur.). Formatex, Badajoz, Spain, 2010, pp.
1355–1366.
[17] Qui Z, Zhao L, Weatherley L. Process intensification technologies in continuous
biodiesel production, Chem Eng Process, 2010;49:323–30.
[18] Behzadi S, Farid MM. Production of biodiesel using a continuous gas-liquid reactor,
Bioresour Technol, 2009;100:683–9.
[19] Sun J, Ju J, Ji L, Zhang L, Xu Z. Synthesis of biodiesel in capillary microreactors, Ind
Eng Chem Res, 2008;47:1398–03.
[20] Budžaki S, Miljić G, Tišma M, Sundaram S, Hessel V. Is there a future for enzymatic
biodiesel industrial production in microreactors? Appl Energy 2017;201:124–34.
[21] Saka S, Kusdiana D. Biodiesel fuel from rapeseed oil as prepared in supercritical
methanol, Fuel, 2001;80:225–31.
[22] Šalić A, Zelić B. Microreactors-portable factories for biodiesel fuel production, Goriva i
maziva, 2011;50:85–110.

T
[23] Ehrfeld W, Hessel V, Löwe H. Microreactors: New technology for modern chemistry,

IP
Wiley-VCH, Weinheim, 2000, pp 1–12.

R
[24] Xie T, Zhang L, Xu N. Biodiesel synthesis in microreactors, Green Process Synth,
2012;1:61–70.

SC
[25] Ognjanović N, Bezbradica D, Knežević Z. Optimization of the production of biodiesel by
commercial immobilized lipaza in a solvent-free system using a response surface
methodology, J Serb Chem Soc, 2008;73:147–56.
U
N
[26] Shah S, Sharma S, Gupta MN. Enzymatic transesterification for biodiesel production,
Indian J Biochem Biophys, 2003;40:392–9.
A
[27] Budžaki S, Šalić A, Zelić B, Tišma M. Enzyme-catalysed biodiesel production from
M

edible and waste cooking oils, Chem Biochem Eng Q, 2015;29:465–9.

[28] Brzozowski AM, Savage H, Verma CS, Turkenburg JP, Lawson DM, Svendsen A,
ED

Patkar S. Structural Origins of the Interfacial Activation in Thermomyces (Humicola)

lanuginosa Lipase, Biochemistry-US 2000;39:15071–82.
PT

[29] Bajaj A, Lohan P, Jha P N, Mehrotra R. Biodiesel production through lipase catalysed
transesterification: An overview, J Mol Catal B: Enzym, 2010;62:9–14.
[30] Akoh CC, Chang SW, Lee GC, Shaw JF. Enzymatic approach to biodiesel production, J.
E

Agric Food Chem, 2007;55:8995–9005.

CC

[31] Aarthy M, Saravanan P, Gowthaman MK, Rose C, Kamini NR. Enzymatic

transesterification for production of biodiesel using yeast lipases: An overview, Chem Eng
A

Res Des, 2014;92:1591–601.

[32] Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, Tominaga Y.
Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase, J Am
Oil Chem Soc, 1999;76(7):789–93.
[33] Fernandez-Lafuente R. Lipase from Thermomyces lanuginosus: Uses and prospects as an
industrial biocatalyst, J Mol Catal B: Enzym, 2010;62:197–212.
[34] Shimada Y, Watanabe Y, Sugihara A., Tominaga T. Review: enzymatic alcoholysis for
biodiesel fuel production and application of the reaction to oil processing, J Mol Catal B:
Enzym, 2002;17:133–42.
[35] Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, et al. Pretreatment of
immobilized Candida antarctica lipase for biodiesel fuel production from plant oil, J Biosci
Bioeng, 2000;90(2):180–3.
[36] Du W, Xu YY, Liu DH, Li ZB. Study on acyl migration in immobilized lipozyme TL
catalyzed transesterification of soybean oil for biodiesel production, J Mol Catal B: Enzym,

T
2005;37:68–71.

IP
[37] Fernandez-Lafuente R. Lipase from Thermomyces lanuginosus: Uses and prospects as an

R
industrial biocatalyst, J Mol Catal B: Enzym, 2010;62:197–212.
[38] Mukherjeea J, Guptab MN. Dual bioimprinting of Thermomyces lanuginosus lipase for

SC
synthesis of biodiesel, Biotechnol Rep, 2016;10:38–43.
[39] Tacias-Pascacio VG, Virgen-Ortíz JJ, Jiménez-Pérez M, Yates M, Torrestiana-Sanchez

U
B, et al. Evaluation of different lipase biocatalysts in the production of biodiesel from used
N
cooking oil: Critical role of the immobilization support, Fuel, 2017;200:1–10.
[40] Du W, Xu Y, Liu D. Lipase-catalysed transesterification of soya bean oil for biodiesel
A
production during continuous batch operation, Biotechnol Appl Biochem, 2003;38:103–6.
M

[41] Silva JA, Macedo GP, Rodrigues DS, Giordano RLC, Gonçalves LRB. Immobilization
of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using
ED

different support activation strategies. Biochem Eng J, 2012;60:16–24.

[42] Verdasco-Martín CM, Villalba M, dos Santos JCS, Tobajas M, Fernandez-Lafuente R,
PT

Otero C. Effect of chemical modification of Novozym 435 on its performance in the

alcoholysis of camelina oil, Biochem Eng J, 2016;111:75–86.
[43] Villalba M, Verdasco-Martín CM, dos Santos JCS, Fernandez-Lafuente R, Otero C.
E

Operational stabilities of different chemical derivatives of Novozym 435 in an alcoholysis

CC

reaction, Enzyme Microb Technol, 2016;90:35–44.

[44] Modi MK, Reddy JRC, Rao BVSK, Prasad RBN. Lipase-mediated conversion of
A

vegetable oils into biodiesel using ethyl acetate as acyl acceptor, Bioresour Technol,
2007;98:1260–4.
[45] Shimada Y, Watanabe Y, Sugihara A, Tominaga Y. Enzymatic alcoholysis for biodiesel
fuel production and application of the reaction to oil processing, J Mol Catal B: Enzym,
2002;17:133–42.
[46] Watanabe Y, Shimada Y, Sugihara A, Noda H, Fukuda H, Tominaga Y. Continuous
production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase,
J Am Oil Chem Soc, 2000;77:355–60.
[47] Zhao X, El-Zahab B, Brosnahan R, Perry J, Wang P. An organic soluble lipase for water-
free synthesis of biodiesel, Appl Biochem Biotechnol, 2007;143:236–43.
[48] Shah S, Sharma S, Gupta MN. Biodiesel preparation by lipase-catalyzed
transesterification of jatropha oil, Energy Fuels, 2004;18:154–9.
[49] Cesarini S, Pastor F, Nielsen P, Diaz P. Moving towards a competitive fully enzymatic

T
biodiesel process, Sustainability 2015;7:7884–903.

IP
[50] López-Serrano P, Cao L, Van Rantwijk F, Sheldon RA. Cross-linked enzyme aggregates

R
with enhanced activity: Application to lipases, Biotechnol Lett, 2002;24:1379–83.
[51] Gupta P, Dutt K, Misra S, Raghuwanshi S, Saxena RK. Characterization of cross-linked

SC
immobilized lipase from thermophilic mould thermomyces lanuginosa using glutaraldehyde
Bioresour Technol, 2009;100:4074–76.

U
[52] Charusheela A, Arvind L. Enzyme catalyzed hydrolysis of esters using reversibly soluble
N
polymer conjugated lipases, Enzyme Microb Technol, 2002;30:19–25.
[53] Schultz N, Hobley TJ, Syldatk C. Spectrophotometric assay for online measurement of
A
the activity of lipase immobilised on micro-magnetic particles, Biotechnol Lett, 2007;29:365–
M

71.
[54] Brennan JL, Hatzakis NS, Tshikhudo TR, Dirvianskyte N, Razumas V, et al.
ED

Bionanoconjugation via click chemistry: The creation of functional hybrids of lipases and
gold nanoparticles, Bioconjugate Chem 2006;17:1373–75.
PT

[55] Betancor L, Fuentes M, Dellamora-Ortiz G, López-Gallego F, Hidalgo Aet al. Dextran

aldehyde coating of glucose oxidase immobilized on magnetic nanoparticles prevents its
inactivation by gas bubbles, J Mol Catal B: Enzym, 2005;32:97–101.
E

[56] Rodrigues RC, Godoy CA, Filice M, Bolivar JM, Palau-Ors A, et al.Reactivation of
CC

covalently immobilized lipase from Thermomyces lanuginosus, Process Biochem,

2009;44:641–6.
A

[57] ASTM D6751-15ce1, Standard Specification for Biodiesel Fuel Blend Stock (B100) for
Middle Distillate Fuels, ASTM International, West Conshohocken, PA, 2015.
[58] DIN EN 14214, Liquid petroleum products - Fatty acid methyl esters (FAME) for use in
diesel engines and heating applications - Requirements and test methods (includes
Amendment A1:2014), EU, 2015.
[59] Atadashi IM, Aroua MK, Abdul Aziz A. Biodiesel separation and purification: A review,
Renewable Energy, 2011;36:437–43.
[60] Ho KC, Shahbaz K, Rashmi W, Mjalli FS, Hashim MA, Alnashef IM, Removal of
glycerol from palm oil-based biodiesel using new ionic liquids analogues, J Eng Sci Technol,
2015;14:98–111.
[61] Abbott AP, Capper G, Davies DL, Rasheed RK, Tambyrajah V. Novel solvent properties
of coholine chloride/urea mixtures, Chem Commun, 2003;1:70–71.
[62] Shahbaz K, Mjalli FS, Hashim MA, Al Nashef IM. Eutectic solvents for the removal of

T
residual palm oil-based biodiesel catalyst, Sep Purif Technol, 2011;81:216–22.

IP
[63] Shahbaz K, Mjalli FS, Hashim MA, Al Nashef IM. Elimination of all free glycerol and

R
reduction of total glycerol from palm oil-based biodiesel using non-glycerol based deep
eutective solvents, Sep Sci Technol, 2013;48:1184–93.

SC
[64] Tang B, Lee YL, Row KH. Exploration of using deep eutectic solvents to separate
methyl palmitate from simulated biodiesel mixtures, Adv Mater Res, 2015;1101:249–51.

U
[65] Šalić A, Pindrić K, Zelić B. Bioproduction of food additives hexanal and hexanoic acid
N
in a microreactor, Appl Biochem Biotechnol, 2013;171:2273–84.
[66] Šalić A, Pindrić K, Hojnik Podrepšek G, Leitgeb M, Zelić B. NADH oxidation in a
A
microreactor catalysed by ADH immobilised on γ-Fe2O3 nanoparticles, Green Process Synth,
M

2013;2:569–78.
[67] Mogensen JE, Sehgal P, Otzen DE. Activation, inhibition, and destabilization of
ED

Thermomyces lanuginosus lipase by detergents, Biochemistry – US, 2005;44:1719–30.

[68] Martinelle M, Holmquist M, Hult K. On the interfacial activation of Candida antarctica
PT

lipase A and B as compared with Humicola lanuginosa lipase, Biochim Biophys Acta,
1995;1258:272–6.
[69] Jutila A, Zhu K, Patkar SA, Vind J, Svendsen A, Kinnunen PK. Detergent-induced
E

conformational changes of Humicola lanuginosa lipase studied by fluorescence spectroscopy,

CC

Biophys J, 2000;78:1634–42.
[70] Sörensen MH, Ng JBS, Bergström L, Alberius PCA. Improved enzymatic activity of
A

Thermomyces lanuginosus lipase immobilized in a hydrophobic particulate mesoporous

carrier, J Colloid Interface Sci, 2010;343:359–65.
[71] Palacios D, Busto MD, Ortega N. Study of a new spectrophotometric end-point assay for
lipase activity determination in aqueous media, LWT Food Sci Technol, 2014;55:536–42.
[72] Pöhnlein M, Finkbeiner T, Syldatk C, Hausmann R. Development of a microtiter plate-
based assay for the detection of lipase-catalyzed transesterification in organic solvents,
Biotechnol Lett, 2015;37:705–10.
[73] Marmur J. A procedure for the isolation of deoxyribonucleic acid from microorganisms,
J Mol Biol, 1961;3:208–18.
[74] Invernizzi G, Casiraghi L, Grandori R, Lotti M. Deactivation and unfolding are
uncoupled in a bacterial lipase exposed to heat, low pH and organic solvents, J Biotechnol,
2009;141:42–6.

T
[75] Kamal MdZ, Yedavalli P, Deshmukh MV, Rao NM. Lipase in aqueous-polar organic

IP
solvents: Activity, structure, and stability, Protein Sci, 2013;22:904–15.

R
[76] Yeh SI, Huang YC, Cheng CH, Cheng CM, Yang JT. Development of a millimetrically
scaled biodiesel transesterification device that relies on a droplet-based co-axial fluids, Sci

SC
Rep, 2016;6:1–7.
[77] Lu J, Chen Y, Wang F, Tan T. Effect of water on methanolysis of glycerol trioleate

U
catalysed by immobilized lipase Candida sp. 99-125 in organic solvent system, J Mol Catal B:
N
Enzym, 2009;56:122–5.
[78] Robles-Medina A, Gonzalez-Moreno PA, Esteban-Cerdán L, Molina-Grima E.
A
Biocatalysis: Towards ever greener biodiesel production, Biotechnol Adv, 2009;27:398–408.
M

[79] Pongket U, Piyatheerawong W, Thapphasaraphong S, H-Kittikun A. Enzymatic

preparation of linoleic acid from sunflower oil: an experimental design approach, Biotechnol
ED

Biotechnol Equip, 2015;29 926–34.

[80] Ghaly AE, Dave D, Brooks MS, Budge S. Production of biodiesel by enzymatic
PT

transesterification: Review, Am J Biochem Biotechnol, 2010;6:54–76.

[81] Patil PD, Gude VG, Reddy HK, Muppaneni T, Deng S. Biodiesel production from waste
cooking oil using sulphuric acid and microwave irradiation processes, J Environ Prot,
E

2012;3:107–13.
CC

[82] Sahoo HR, Kralj JG, Jensen KF. Multistep continuous-flow microchemical synthesis
involving multiple reactions and separations, Angew Chem Int Ed, 2007;46:5704–8.
A

[83] Baxendale IR, Deeley J, Griffiths-Jones CM, Ley SV, Saaby S, Tranmer GK. A flow
process for the multi-step synthesis of the alkaloid natural product oxomaritidine: A new
paradigm for molecular assembly, Chem Commun, 2006;24:2566–658.
[84] Sun PY. Preparation and purification of biodiesel in microstructured reactors, PhD
dissertation, Nanjing University of Technology, Nanjing, 2010.
List of figures:
Figure 1. Process scheme for biodiesel synthesis in a microreactor

Figure 2. Process scheme for biodiesel production and purification with two microchips
connected in series (biodiesel production in the first chip, biodiesel purification in the second
chip)

Figure 3. Effect of emulsifier on dynamic of phase separation for a) water:oil and b)

alcohol:oil system

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Figure 4. (a) Effect of different organic solvents on enzyme activity with (b) dynamics of
enzyme deactivation under the influence of Marmur solution and (c) the effect of thermal

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deactivation coupled with Marmur solution

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Figure 5. Enzyme deactivation in the presence of methanol

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Figure 6. Biodiesel production in a microreactor: (a) influence of feeding and temperature (●
oil introduced as separate phase, ● enzyme introduced as separate phase, ○ methanol
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introduced as separate phase) and (b) influence of oil and temperature (∆ reaction performed
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with waste cooking oil, □ reaction performed at room temperature with sun flower oil (21
M

°C))
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