J Am Oil Chem Soc (2016) 93:905–910
DOI 10.1007/s11746-016-2843-4
ORIGINAL PAPER
Production of Biodiesel Using Liquid Lipase Formulations
P. M. Nielsen1 · A. Rancke‑Madsen1 · H. C. Holm1 · R. Burton2 
Received: 9 September 2015 / Revised: 29 April 2016 / Accepted: 29 April 2016 / Published online: 13 May 2016
© AOCS 2016
Abstract  Looking back at the literature for enzymatic bio-
diesel, it is evident that the research has been focused on
using immobilized lipase to enable re-use of the enzyme
due to price constraints on lipases used for catalyzing the
transesterification process. The use of liquid formulations
of lipase for biodiesel has recently been implemented in the
industry. Technology for using liquid formulated lipases for
enzymatic biodiesel production is new and, since enzyme
prices have been reduced, it is now possible to simplify
the process considerably and apply it for very low-quality
oils. In this paper, the use of liquid lipase formulations for
enzymatic biodiesel will be described along with a general
proposal for an industrial-scale enzymatic biodiesel process
with >95 % yield.
Keywords  Biodiesel; Biobased poducts · Biofuels
(Energy); Biobased products · Enzymology; Biotechnology
biocatalysis · Fats and oils · Esterification; Processing
technology
Introduction
Recently, a comprehensive article reviewing the aspects of
using immobilized lipases for enzymatic biodiesel was pub-
lished [1]. Due to the economics of producing the immobi-
lized product, the reuse of the enzyme is of key importance,
but with several complications when considering a re-use
strategy [2, 3]. Stability of the enzyme can be a challenge
* P. M. Nielsen
pmn@novozymes.com
1
Novozymes A/S Bagsvaerd, Copenhagen, Denmark
2
Novozymes A/S Bagsvaerd, Franklington, USA
both due to the risk of enzyme leaching from the carrier
and shear stress breaking carrier into smaller particles.
Also, the risk of enzyme deactivation caused by the alco-
hol, and accumulation of glycerin on the carrier particles
is emphasized [4]. The reactor construction must be care-
fully designed to make sure the mass transfer is high with-
out compromising the mechanical stability of the enzyme
carrier. The type of reactor for immobilized lipase has been
extensively discussed and includes suggestions of mixed-
batch tanks, air-lifted reactors, packed-bed reactors and
expanded-bed reactors [5]. Each of these reactor types has
limitations related to the enzyme formulation. The mixed-
batch reactors cannot be equipped with an efficient mixer,
since high shear rate tends to break down the relatively
fragile carrier. That is why a gentle way of mixing using air
bubbles has been suggested. For the packed-bed operation,
there is another issue where the glycerin produced from the
reaction can accumulate in the bed and result in a reduced
reaction rate as well as an increased pressure drop over the
column [6]. That has led to the expanded bed system with
up-flow to introduce some movement of the enzyme parti-
cles in order to prevent glycerin attachment to the enzyme
particles.
The use of liquid formulated lipases eliminates several
of the problems discussed above but has been described in
a few articles only [7–12], and show how a simple well
mixed reactor can be used for biodiesel production using
liquid lipase as the catalyst. The articles by Cesarini et al.
[7], and Lv et al. [8] discuss reaction mechanistic details
when using liquid enzyme for fatty acid methyl esters
(FAME), and show how the enzyme initiates the reac-
tion by producing a significant increase in free fatty acids
(FFA) concentration followed by esterification. A clear
correlation between the initial water concentration and
the resulting FFA was documented [7]. Soybean oil was
13

906
reacted with 15 % methanol at 35 °C for 24 h using 1 %
w/w of oil Callera Trans (Novozymes, Bagsvaerd, Den-
mark) and with varying content of water from 3 % w/w
of oil + water to 15 %. For low water addition the result-
ing FFA was 2.5 % w/w of dry FAME phase. At 5, 10
and 15 % the FFA was 2.8, 3.8 and 5.3 %, respectively.
Price et al. [14] modeled the reaction rate for liquid lipase
catalyzed transesterification to optimize the process with
respect to methanol dosage, water, and enzyme concentra-
tion. In a later publication [15] a work with the data from
biodiesel production in laboratory, pilot, and full scale
productions showed it was possible to develop reliable
modeling of the process and with simple control analysis.
Depending upon the type of feedstock (high or low free
fatty acids content) it was found that the reaction could be
controlled by analysis the FFA content in Brown Grease
having a high FFA from the start. The used cooking oil
transesterification could be controlled by analyzing the
content of bound glycerin.
The enzyme dosage is very important for the total cost of
the process and a good compromise between enzyme dos-
age and reaction time has been established. The study by
Nordblad et al. shows the reaction rate of transesterification
depends upon temperature and enzyme dosage when using
Eversa Transform (Novozymes, Denmark). Increasing the
enzyme dosage from 0.1 to 0.2 % in a 35 °C reaction mix-
ture increased the reaction rate by 65 %, while going from
0.2 to 0.3 % resulted in a marginal 16 % increase in the
reaction rate.
Surprisingly, it is possible to recover the liquid enzyme
in a simple manner [12]. The enzyme is an amphiphilic
molecule working on the interface between water and oil.
This means that the enzyme is concentrated in an emulsion
phase between a clear heavy phase and the FAME phase
after separation by gravity. It has been found that 95 %
of the enzyme activity is located in this emulsion phase
(un-published data). However, recent advances have now
reduced the enzyme price to a level where re-use of the
enzyme is unnecessary, and a one-time use of the enzyme
is economically viable. Being able to discharge the enzyme
after only one use eliminates the risk of accumulating com-
ponents coming in with the oil that can reduce the separa-
tion efficiency and also resulting in a simpler, more repro-
ducible process.
The articles discussing enzyme catalyzed transesterifica-
tion reactions are in fact normally not leading to a final bio-
diesel fulfilling the specifications for biodiesel (e.g., acid
value max. 0.5mg KOH/g corresponding to FFA <0.25 %,
and total glycerol ≤0.24 % as stated in the ASTM stand-
ard),1 although they describe an efficient process leading to
1
  ASTM standard D6751 United States.
13
J Am Oil Chem Soc (2016) 93:905–910
a high yield of esters. There still remains a low level of
FFA that needs to be further reduced to below 0.25 %.
The latest research and development has resulted in a
simple full process for enzymatic biodiesel consisting of
two steps. The first step is the transesterification catalyzed
by liquid lipase, and the second step is a polishing process
where the biodiesel is brought into end-product specifica-
tions by reducing the FFA to less than 0.25 %. We describe
two alternatives for polishing: esterification of residual FFA
by enzyme catalyzed reaction, and caustic wash of FFA to
produce soap followed by centrifugation.
In this paper we document how an enzymatic transes-
terification can be made using a liquid lipase formulation
which circumvents a lot of the process issues related to the
use of immobilized lipase. Furthermore, a simple down-
stream process needed for producing biodiesel within spec-
ifications is documented.
Experimental Procedures
The transesterification reaction mixture comprised refined
soybean oil, 0.2 w/w% Eversa Transform (Novozymes,
Denmark), 3 w/w% water, and 1.5 molar equivalents
(equiv) of methanol (based on feedstock fatty acids). Meth-
anol was added during the reaction by dosing 0.2 equiv at
t  = 0 and additionally dosing 0.06 equiv/h. The reaction
was conducted in a 1 L temperature controlled glass reac-
tor with three baffles, and mixed with an IKA propeller
mixer with a stirrer rate of 500 rpm at 35 °C. The height
of the reactor was 14 cm and the diameter 10 cm. During
this reaction the crude FAME produced contained 1.13 %
MAG, 0.32 % DAG, 0.11 % TAG, and 1.2 % FFA.
Materials used in the experiments were enzymes: Eversa
Transform Lipase, 100 kLU/g (kLU = 1000 LU. 1 LU is
the amount of enzyme that realeases 1 µmol of titratable
butyric acid per min at pH = 7.00 and 35 °C) (Novozymes,
bagsvaerd, Denmark. Lipozyme CalB L, 5kLU/g (Novo-
zymes, Denmark).
Other Materials
Methanol and glycerin both HPLC grade >99.5 % purity
(Sigma Aldrich, Denmark). Refined two setups were used
for the esterification trials using a liquid enzyme. In the
first experiment to 40 g of biodiesel was added oleic acid
to 2.1 % FFA, 1 wt% (0.4 g) Lipozyme CALB-L (Novo-
zymes, Denmark), 2.57 % (1.026 g) methanol, and 10 %
ACS grade glycerol were combined in a 120-mL bot-
tle. An air flow rate of 617 mL/min was applied and re-
circulated in a 20-mL methanol column. Results from
this trial are seen in Fig. 3. The other esterification reac-
tion (polishing) was made in the same closed mixed 1-L
J Am Oil Chem Soc (2016) 93:905–910 907
Fig. 1  Transesterification
process during the first 24 h of
reaction. 0.2 % Eversa Trans-
form lipase, 1.5 mol equivalent
methanol added over 22 h. Tem-
perature 35 °C. + FAME, filled
diamonds FFA, filled triangles
TAG, filled circles DAG, filled
squares MAG
reactor used for the transesterification with a stirrer oper-
ated at 500 rpm at 45 °C. The stirrer was an IKA Euro-ST
D, 75 W, 50–200 rpm from IKA Werke, Staufen, Germany.
The reactor was loaded with crude FAME with FFA, liq-
uid enzyme CalB (Novozymes) at a dosage of 0.4 % w/w
on FAME, and 3 % glycerin. In the case of running the
esterification directly after transesterification, the added
glycerin can be omitted. The whole system is flushed
with nitrogen before starting. From the headspace of the
reactor, air was pumped to a condenser at a rate of 0.8 L
air/min (0.8VVM). The pump was a F9 V vacuum pump
from Fürgut GmbH, Tannheim, Germany. The condenser
consisted of a methanol reservoir with an air sparger that
distributed the air in small bubbles. The temperature of
the condenser was 25 °C. A second F9 V vacuum pump
pulled dry methanol/nitrogen from the headspace of the
methanol condenser to feed the reactor. This air loop reac-
tor is described in a patent application [13]. This reactor
system is capable of keeping the methanol concentra-
tion at approximately 2 % in the reactor and the water at
400 ppm.
The polishing process using caustic addition to the total
reaction mixture from transesterification was tested by add-
ing NaOH (1.15 mol excess to FFA, added as a 4 % solu-
tion) to the crude FAME at 60 °C. After a 30-min reaction
time, the mixture separated into a heavy and a light phase.
This process is called a one-pot process as it can be carried
out in the transesterification reaction tank. Determination
of yield for the one-pot process was made by calculating
the weight of dry biodiesel relative to the weight of feed-
stock used in transesterification.
The analytical techniques used were as follows. FFA
was analyzed by titration according to the AOCS method
(Ca 5a-40).2 Water content was analyzed by Karl Fischer
titration. Methanol and glycerides were analyzed using the
QTA Medium Infrared System supported by the Eurofins
calibration service according to AOCS specification.3 The
bound glycerin (BG) is the total amount of glycerin bound
in the glycerides, and is calculated from knowing the con-
tent of glycerides.
Results and Discussion
One of the strategies we have been exploring for bringing
down the cost of enzyme in the transesterification process
has been to re-use the enzyme. It was found that the lipase
being a protein molecule which is trying to position on the
interface between water and oil stays in an emulsion phase
after the reaction mixture is allowed to settle. In practice
the separation process normally produces a clear FAME
phase, but it can be difficult to obtain the clear glycerin
phase and recover the relatively small emulsion layer.
The difficulty with recovering the small enzyme emul-
sion layer has led to a strategy where the separation left the
glycerin/emulsion phase in the reactor for the next batches.
With this new strategy, the enzyme loss is small but the
amount of glycerin phase builds up from batch to batch,
limiting enzyme usage to only 3–4 batches.
The enzyme price has been reduced so much lately
that it pays off to consider one-time enzyme usage. In the
2
  AOCS Ca 5a-40. Free fatty acids.
3
 AOCS Ck 2-09 standard method for method for the analysis of
methanol content, cloud point, monoglyceride, total and free glycerin
in biodiesel.
13

908 J Am Oil Chem Soc (2016) 93:905–910

1.6 1.6
y = 0.0006x - 0.0932
1.4 1.4
R² = 0.9882
1.2 1.2
1 1
FFA, %

0.8 0.8

%
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 500 1000 1500 2000 2500 0 5 10 15 20
Water, ppm Reacon me, hr

Fig. 2  Equilibrium between water and FFA after esterification with
CalB at different water contents. R2 = 0.998. Samples taken from the
reaction mixture when equilibrium was established at different water
contents
Fig. 4  Esterification of crude FAME in an air-stripped mixed reac-
tor at 45 °C using 0.4 % liquid CalB and 4 % glycerin. Air flow rate
0.08VVM with a propeller mixer in the reactor operating at 500 rpm
filled diamonds FFA, filled squares MAG, filled triangles bound glyc-
erin (BG)

2.5
FFA which needs to be eliminated to pass the quality speci-
2 fication of <0.25 % FFA.
There are several options to reduce the residual FFA
1.5
in crude FAME after methanol recovery. One option is to
saponify the FFA in the FAME by washing with a caustic
FFA, %

solution followed by separation. This has shown in prac-

1
y = 2.2865e-0.011x
0.5 R² = 0.9986
0.25%
0 and optimized caustic wash.
0 50 100 150 200
Reacon me, minutes
Fig. 3  Data from the esterification reaction using liquid CalB to
reduce FFA in 40 g FAME with 2.1 % FFA and with 10 % glycerin
added. Enzyme dosage 1 % Lipozyme CalB (Novozymes) of oil
phase. 35 °C reactor temperature. Air flow rate 617 mL/min
one-time use approach, a low dosage of Eversa Transform
(0.2 w/w on oil) is used and the reaction time is prolonged
to 35–36 h to reach equilibrium for the transesterification.
Typical changes in composition of FAME, glycerides, and
FFA during the first 24-h transesterification reaction are
shown in Fig. 1. It is clearly seen how FFA and di-glycer-
ides (DAG) increase in the first hours of the reaction. The
reaction with low enzyme dosage of 0.2 % typically takes
32–36 h to reach equilibrium. With one-time use of the
enzyme it is now possible to heat up the reaction mixture to
achieve a better phase separation.
The transesterification process based on liquid formu-
lated lipases has been undertaken in large scale operation
for some years now in fed-batch [15] as well as in continu-
ous stirred tank reactors and shown to work well on many
different qualities of oil. The process leaves typically 2 %
tice to be troublesome due to the solubility of soap in the
FAME leading to difficulties in soap separation. Alterna-
tives that have been explored further in this study include
esterification of the remaining FFA by enzymatic catalysis
250 Enzymatic catalyzed esterification was shown to be
feasible already some years ago [13]. In this setup it was
possible to eliminate the water produced by esterification
and keep the water content at 500 ppm which is required
to arrive at <0.25 % FFA at the end of the reaction. Man-
aging the water content in the reactor is the key to control
the FFA content in an esterification reaction as is shown on
Fig.  2. These data were obtained by reacting FAME with
different FFA contents to equilibrium and then measur-
ing the water and FFA after the reaction. Each 1 % FFA
esterified, produces 650 ppm water. High enzyme dosage
and high air flow rate were used and that needed to be opti-
mized to be economical feasible in large scale. Figure 3
shows data from the esterification of crude FAME from
the transesterification process after the emulsion and heavy
phase has been eliminated. The FAME containing 2.2 %
FFA was reacted for 200 min using 1 % liquid CalB, which
reduced the FFA to <0.25 %.
The data shown in Fig. 4 demonstrate an FFA con-
tent  <0.25 % with 10 % of the original air flow and
enzyme dosage reduced to 0.4 %. Furthermore, we also
converted some of the remaining glycerides to FAME
which improves the quality of the FAME and also adds to
increased yield. The mixer mounted in the reactor secured

13
J Am Oil Chem Soc (2016) 93:905–910 909
1 remains is to have a continuous production experience to
0.9
0.8
0.7
0.6
0.5
%
0.4
0.3
0.2
0.1
0
0 0.2 0.4 0.6 0.8 1 1.2
Reacon me, hr
Fig. 5  Data from the caustic wash “one-pot” reaction to bring FFA
and BG below specification limits. Conditions: crude FAME with
0.8 % FFA was added 1.15 mol NaOH to mol FFA as a 4 % solution
in water, 60 °C, 500 rpm, 1 h. Reaction mixture was separated before
analysis. filled diamonds FFA, filled squares MAG, filled triangles
BG
a very good mass transfer which is why the air flow could
be dramatically reduced. Figure 4 shows the composition
of the FAME phase during the reaction of crude FAME at
45 °C with optimized parameters with respect to enzyme
dosage (0.4 % w/w on oil of CalB) and air flow rate
(0.08VVM). Reduction in FFA to <0.25 % took 17 h and at
the same time MAG and TAG were significantly reduced.
The enzymatic catalyzed esterification process produces
FAME from the remaining FFA and also transesterify a part
of the glycerides which is desirable. The drawback with
this process is that it has not yet been scaled up to prove the
concept and will require some new reactor technology to
make it efficient.
Alternatively to the enzymatic esterification processes
for polishing we have worked with a new and simpler way
of doing caustic wash [16]. The process utilizes the whole
reaction mixture from the transesterification process still
with the excess methanol present and results in an efficient
separation process after the soap formation. The use of
NaOH surplus to saponify the remaining FFA was tested.
Figure 5 shows data from caustic washing. The experiment
was carried out in the laboratory 1-L reactor as described
above. Reduction in FFA to <0.25 % took a maximum of
30 min, BG is reduced to <0.22 %, and at the same time
MAG was reduced from 0.9 to 0.6 %. After the reaction,
the phase separated easily due to the absence of an emul-
sion phase between the biodiesel phase and the heavy
phase. The yield obtained was 97.5 % w/w.
Experiences from full scale production show that the
one-pot-polishing is a robust process with a high yield. That
makes it the preferred process for reducing FFA to <0.25 %
after enzymatic transesterification. Both of the two alterna-
tive polishing processes (enzymatic esterification and the
one-pot polishing) are now operated on a full scale. What
be able to provide the data that can be used to document the
full process economy.
Conclusion
A cost efficient method for the production of biodiesel
using enzymatic catalyzed transesterification is proposed.
The first step of the process is transesterification catalyzed
by a liquid lipase formulation. After this, the remaining
FFA is reduced to <0.25 % by a saponification process
(one-pot-process) carried out on the total reaction mixture
from the transesterification. The overall yield shown from
full scale productions is >97 %. The one-pot-polishing
process has already been scaled up to production scale
operation.
References
1. Xuebin Z, Feng Q, Chongli Y, Wei D, Dehua L (2015) Lipase-
catalyzed process for biodiesel production: enzyme immobiliza-
tion, process simulation and optimization. Renew Sustain Energy
Rev 44:182–197
2. Fjerbaek L, Christensen KV, Norddahl B (2009) A review of the
current state of biodiesel production using enzymatic transesteri-
fication. Biotechnol Bioeng 102:1298–1315
3. Nielsen PM, Brask J, Fjerbaek L (2008) Enzymatic biodiesel
production: technical and economical considerations. Eur J Lipid
Sci Technol 110:692–700
4. Xu Y, Nordblad M, Nielsen PM, Brask J, Woodley JM (2011)
In situ visualization and effect of glycerol in lipase-catalyzed
ethanolysis of rapeseed oil. J Mol Catal B Enzym 72:213–219
5. Poppe JK, Fernandez-Lafuente R, Rodrigues RC, Ayub MAZ
(2015) Enzymatic reactors for biodiesel synthesis: present status
and future prospects. Biotech Adv 33:511–525
6. Xu Y, Nordblad M, Nielsen PM, Brask J, Woodly JM (2011)
In situ visualization and effect of glycerol in lipase-catalyzed
ethanolysis of rapeseed oil. J Mol Catal B Enzym 72:213–219
7. Cesarini S, Diaz P, Nielsen PM (2013) Short communication:
exploring a new, soluble lipase for FAMEs production in water-
containing systems using crude soybean oil as a feedstock. Pro-
cess Biochem 48:484–487
8. Lv D, Du W, Zhang G, Liu D (2010) Mechanism study on
NS81006-mediated methanolysis of triglyceride in oil/water
biphasic system for biodiesel production. Process Biochem
45:446–450
9. Kaied M, Samukawa T, Kondo A, Fukuda H (2001) Effect of
methanol and water contents on production of biodiesel fuel
from plant oil catalyzed by various lipases in a solvent-free sys-
tem. J Biosci Bioeng 91:12–15
10. Ren H, Li Y, Du W, Liu D (2013) Free lipase-catalyzed esteri-
fication of oleic acid for fatty acid ethyl ester preparation with
response surface optimization. J Am Oil Chem Soc 90:73–79
11. Nordblad M, Silva VTL, Woodley JM, Nielsen PM (2014) Iden-
tification of critical parameters in liquid enzyme-catalyzed bio-
diesel production. Biotechnol Bioeng 111(12):2446–2453
12. Pedersen AT, Nordblad M, Nielsen PM, Woodley JM (2014)
Batch production of faee-biodiesel using a liquid lipase formula-
tion. J Mol Catal B Enzym 105:89–94
13

910 J Am Oil Chem Soc (2016) 93:905–910

13. Austin G, Burton R, Fan X (2012) Patent application WO

to 40 m3 using a liquid lipase formulation. Biotech Bioeng
2012/106701. Fatty Acid Esterification Process (accepted. ID 1510-61)
14. Price J, Hofmann B, Silva VTL, Nordblad M, Woodley JM, Huu- 16. Rancke-Madsen A, Nielsen PM. Pat. WO2015/181308. Produc-
som JK (2014) Mechanistic modelling of biodiesel production tion of fatty acid alkyl esters with caustic treatment
using a liquid lipase formulation. Biotechnol Prog 30:1277–1290
15. Price J, Nordblad M, Martel H, Chrabas B, Wang H, Nielsen PM,
Woodley J (2015) Scale-up of industrial biodiesel production

13