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Energy Procedia 00
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(2017)000–000
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4th International Conference on Energy and Environment Research, ICEER 2017, 17-21 July
2017, Porto, Spain

Effect of
Thechitosan’s amino
15th International grouponinDistrict
Symposium adsorption-crosslinking
Heating and Cooling
immobilization of lipase enzyme on resin to catalyze biodiesel
Assessing the feasibility of using the heat demand-outdoor
synthesis
temperature function for a long-term district heat demand forecast
Gandhi Alamsyah, Vania Anisya Albels, Muhammad Sahlan, Heri Hermansyah*
I. Andrića,b,c*, A. Pinaa, P. Ferrãoa, J. Fournierb., B. Lacarrièrec, O. Le Correc
Department of Chemical Engineering, Faculty of Engineering, University of Indonesia, 16424 Depok, Indonesia
a
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
b
Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France
c
Abstract Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France

Lipase as biocatalyst is used in biodiesel production but its price is becoming serious problem. Immobilization could
improve the ability of enzyme. Immobilization method which gives higher activity and stability is adsorption-
Abstract
crosslinking method. The addition of amino group on supports has proven to stabilize the enzyme. Thus, this
research focused on the improvement of lipase immobilization performance by the addition of chitosan which
District heating networks are commonly addressed in the literature as one of the most effective solutions for decreasing the
contains amino group. The highest unit activity (24.69 U/g resin) is reached by immobilized lipase on anion-
greenhouse gas emissions from the building sector. These systems require high investments which are returned through the heat
exchange macroporous resin with addition of chitosan on resin directly. This enzyme produces biodiesel with yield
sales. Due to the changed climate conditions and building renovation policies, heat demand in the future could decrease,
ofprolonging
50.79%. the investment return period.
The main scope of this paper is to assess the feasibility of using the heat demand – outdoor temperature function for heat demand
©forecast.
2017 The Authors.
The districtPublished by Elsevier
of Alvalade, locatedLtd.
in Lisbon (Portugal), was used as a case study. The district is consisted of 665
Peer-review under
buildings that varyresponsibility of the scientific
in both construction periodcommittee of theThree
and typology. 4th International Conference
weather scenarios (low,on Energy and
medium, high)Environment
and three district
Research.
renovation scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained heat demand values were
compared with results from a dynamic heat demand model, previously developed and validated by the authors.
Keywords: Adsorption-crosslinking; chitosan; immobilization; lipase; resin
The results showed that when only weather change is considered, the margin of error could be acceptable for some applications
(the error in annual demand was lower than 20% for all weather scenarios considered). However, after introducing renovation
scenarios, the error value increased up to 59.5% (depending on the weather and renovation scenarios combination considered).
1.The
Introduction
value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the
decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and
Lipase isscenarios
renovation one ofconsidered).
the enzymes which
On the otherused
hand,to synthesis
function biodiesel.
intercept However,
increased this per
for 7.8-12.7% commercial lipase isontoo
decade (depending the
expensive, difficultThe
coupled scenarios). to separate and cannot
values suggested couldbebereused after
used to the reaction.
modify Thus,
the function some immobilization
parameters for the scenariosmethods were
considered, and
improve the accuracy of heat demand estimations.

© 2017 The Authors. Published by Elsevier Ltd.

Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and
Cooling.
* Corresponding author. Tel. and fax: +62-813-8539-9048; +62-217-863-515.
E-mail address: heri@che.ui.ac.id
Keywords: Heat demand; Forecast; Climate change
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 4th International Conference on Energy and Environment
Research.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 4th International Conference on Energy and Environment
Research.
10.1016/j.egypro.2017.10.278
48 Gandhi Alamsyah et al. / Energy Procedia 136 (2017) 47–52
Alamsyah et al./ Energy Procedia 00 (2017) 000–000

developed to put the enzyme in a space physically to retain its catalytic activity [1-3]. The term of space means solid
matrix that is called as support. Enzyme immobilization could separate enzyme from medium, so enzyme could
reuse in many reaction. One of immobilization methods is adsorption-crosslinking that has higher stability and
activity than other crosslinking methods (CLEA and CLEC). Crosslinking reaction is a formation of three
dimensional networks between enzyme, support, and reagent. However, this method is not totally perfect due to the
lack of amino groups on the support, which led a decrease of enzyme activity due to the conformational changes of
enzyme. Some previous researchers [1-6] proposed the addition of chitosan as a source of amino group on the
support in enzyme immobilization. The addition of chitosan is expected to increase stability of enzyme
immobilization on resin. Chitosan addition on enzyme immobilization should be examined further to get chitosan
addition method and types of resin which is resulting excellent enzyme activity. Thus, this research will be focused
on the effect of chitosan’s amino group that are seen from chitosan addition method in commercial lipase enzyme
immobilization on various types of resin on the enzyme activity and immobilized enzyme stability.

2. Methods

2.1. Lipase enzyme preparation

1 g of commercial lipase Candida rugosa powder is dissolved in 10 mL phosphate buffer (pH 7) to obtain lipase
solution 0.01 g/mL.

2.2. Adsorption-crosslinking immobilization

Commercial lipase enzyme will be adsorbed on resin, and then treated with glutaraldehyde as crosslinking
reagent. In the first method (method 1), chitosan solution (0.2 g chitosan powder dissolved in CH 3COOH 5% 25
mL) is adsorbed on resin. Then, the lipase solution is mixed with 0.75 g resin – chitosan and stirred in shaker water
bath with conditions of 30 ºC, 150 rpm for 4 hours. After that, 0.5% w/v 5 mL of glutaraldehyde is added and be
stirred with conditions 30 ºC, 150 rpm for 20 minutes. In the second method (method 2), lipase solution is mixed
with 0.75 g resin and stirred in shaker water bath with conditions 30 ºC, 150 rpm for 24 hours. After that, 1 mL of
chitosan solution (0.5 g chitosan powder dissolved in HCl 0.5% 100 mL) parallel with addition of 1% w/v 1 mL of
glutaraldehyde, and be stirred with conditions 30 ºC, 150 rpm for 20 minutes. Enzyme concentration is measured by
using Lowry method. Enzyme loading is the percentage of lipase concentration of successful immobilized enzyme
for adsorption and crosslinking on resin.

2.3. Hydrolysis reaction of enzyme

In this research, lipase is used as biocatalyst in hydrolysis reaction. Substrate in this reaction is triglyceride from
olive oil. Free fatty acid (FFA) content is measured by titration with NaOH base. 5 mL of olive oil is added to 5 mL
aquades and 0.3 g PVA as emulsifier. Then, 5% wt of immobilized lipase is added to the system. Operating
condition of the reaction is 30 ºC, 150 rpm for 30 minutes. The hydrolysis result is added with ethanol and PP
indicator.

2.4. Enzyme activity test

Immobilized lipase is used as biocatalyst in synthesis of biodiesel non-alcohol route batch reactor. Reactor in this
term is 100 mL Erlenmeyer flask. The reactor contains a mixture of palm oil and methyl acetate with 1:12 mole
ratio. Biocatalyst which used in this test is 4% wt of immobilized enzyme. The operating condition of this reaction is
40 ºC on shaker water bath 150 rpm for 50 hours. The sample results from synthesis of biodiesel is analysed by
HPLC (High Performance Liquid Chromatography) to obtain methyl oleate (biodiesel) concentration that have been
formed. The stability test in this research is using biocatalyst to synthesis the biodiesel repeatedly.
 GandhietAlamsyah
Alamsyah et al.
al./ Energy / Energy
Procedia 00Procedia 136 (2017) 47–52
(2017) 000–000 49

2.5. Immobilization of lipase from Aspergillus niger

After obtaining optimum condition of enzyme immobilization, this condition is applied to immobilize lipase from
Aspergillus niger. This condition means chitosan addition method and types of resin, which resulted highest activity
and stability of enzyme. Lipase from Aspergillus niger is produced based on previous research [7, 8]. Production of
lipase from Aspergillus niger including fermentation process, mixing, extraction, centrifugation, and drying.

3. Results and discussion

3.1. Effect of the types of resin to enzyme loading

In the first method, there is an increase of trend line for adsorption enzyme loading to total enzyme loading. It is
caused by glutaraldehyde succeed binding an enzyme, a chitosan, enzyme-chitosan, and enzyme-support.
Meanwhile, in the second method, there is a decrease of adsorption enzyme loading to total enzyme loading, except
for anion-exchange macroporous resin. As mentioned previously, in the second method, chitosan is added after
lipase adsorption to resin, parallel with the addition of glutaraldehyde. This decline occurred because glutaraldehyde
made chitosan unstable-bond with resin is break and led to enzyme leaching. In addition, there is a possibility that
chitosan is closed with the porous of support. The closured of porous by chitosan aims to prevent leaching of lipase
on support. However, chitosan blocked the way of other lipase to enter into resin. However, the use of anion-
exchange macroporous resin in the second method has increased from adsorption enzyme loading to total enzyme
loading. It is caused by the structure and functional group of the resin. The porous and functional group of anion-
exchange macroporous resin could hold chitosan bonding in porous and resin’s surface. When the crosslinking
happened, glutaraldehyde is not break chitosan bonding, but strengthen the bonding between enzymes. Fig. 1.a and
1.b show that adsorption enzyme loading in enzyme immobilization using the second method is higher than the first
method. In method 1, chitosan is attached on resin which is resulting in slower diffusion of lipase. Meanwhile in
method 2, chitosan is not attached on resin, so lipase could diffused to resin faster than method 1.

Fig. 1. (a) Effect of the types of resin to enzyme loading of commercial immobilized lipase using the first method; (b) Effect of the types of resin
to enzyme loading of commercial immobilized lipase using the second method.

3.2. Effect of chitosan addition methods to unit activity of enzyme

Fig. 2 shows that AM1 and M2 have the highest unit activity with 24.69 U/g support. AM1 is commercial lipase
enzyme immobilized on anion-exchange macroporous using chitosan addition method 1. Meanwhile, M2 is
commercial lipase enzyme immobilized on macroporous resin using chitosan addition method 2. The highest unit
activity of AM1 strengthen the fact that this type of resin produce immobilized lipase which can work better than
50 Gandhi Alamsyah et al. / Energy Procedia 136 (2017) 47–52
Alamsyah et al./ Energy Procedia 00 (2017) 000–000

other resin. Amino group which is attached on functional group and porous on anion-exchange macroporous resin
could make lipase diffused well into support. This structure also makes chitosan become the great bridge for enzyme
and support.
In method 1, the lowest unit activity of enzyme is reached by enzyme immobilized on anion-exchange gelular
resin. This occurred because anion-exchange gelular resin does not have porous so enzyme is only located on the
surface of resin. This lipase bonding is not strong enough to react with triglyceride. Thus, in hydrolysis reaction,
lipase on resin anion-exchange macroporous may leached and cannot catalyzed hydrolysis reaction. M2 sample is
also have highest unit activity of enzyme. Macroporous resin did not have functional group in their structure.
Because of this structure, chitosan act as protector for lipase bonding with resin. This enzyme is kept well and could
react with olive oil. We can say that, functional group could hold catalytic side of enzyme.

Fig. 2. Effect of chitosan addition method to unit activity of enzyme.

3.3. Synthesis of biodiesel using immobilized commercial lipase

Two enzyme samples (AM1&M2) which have the highest unit activity is tested using interesterification reaction
by using them as biocatalysts to synthesize biodiesel. The calculation results show that AM1 succeed synthesize
biodiesel with yield of 50.8%. This percentage is higher than sample M2. This is showing us that immobilized
enzyme on resin – chitosan could work better to synthesize biodiesel. Chitosan attachment on resin could hold
catalytic side of enzyme. In chitosan addition method 2, lipase is put inside the chitosan that protect system of
enzyme and support. This causes lipase is more difficult to react with triglycerides than sample AM1.
60

50
Yield of Biodiesel (%)

40

30

20

10

0
M2 AM1

Sample

Fig. 3. Comparison of biocatalyst activity of commercial immobilized lipase.

 Gandhi Alamsyah et al. / Energy Procedia 136 (2017) 47–52 51
Alamsyah et al./ Energy Procedia 00 (2017) 000–000

AM1 then used as biocatalyst repeatedly to test its stability. Fig. 4 shows the relative activity of enzyme to the
cycles of enzyme used. Percentage of enzyme relative activity is the ratio of initial activity of enzyme and final
activity of enzyme. Decreasing in enzyme activity is caused by the lost of enzyme in storage process. Shown in Fig.
4 below, immobilized enzyme still has 63.94% residual activity in fourth cycle. Thus, it also has proven that enzyme
immobilization using adsorption-crosslinking could produces very stable biocatalyst.

Relative Activity (%)

Cycle

Fig. 4. Stability of commercial lipase immobilized on anion-exchange macroporous using chitosan addition method 1.

3.4. Immobilization of lipase from Aspergillus niger

Shown in Fig. 5a, commercial lipase still has higher unit activity than lipase from Aspergillus niger, but the unit
activity for the immobilized enzyme activity did not differ too much. This shows that lipase from Aspergillus niger
is feasible to use for further research to large scale production. Using the same condition as commercial lipase,
lipase from Aspergillus niger succeed to synthesize biodiesel with yield of 69.31%. This value is higher than yield
of biodiesel that is synthesized by commercial lipase. This shows that with the same method and condition,
immobilized lipase from Aspergillus niger is as good as commercial lipase that use as biocatalyst for biodiesel
process. The comparison of commercial lipase and lipase from Aspergillus niger activities is shown in Fig. 5b.

a b

Fig. 5. (a) Unit activity comparison of immobilized commercial lipase and lipase from Aspergillus niger; (b) Activity comparison of immobilized
commercial lipase and lipase from Aspergillus niger.
52 Gandhi Alamsyah et al. / Energy Procedia 136 (2017) 47–52
Alamsyah et al./ Energy Procedia 00 (2017) 000–000

Lipase from Aspergillus niger immobilized on anion-exchange macroporous resin using method 1 is used
repeatedly to test their stability. The results show that lipase from Aspergillus niger still has 70.61% residual
activity. It also shows that the decreasing of enzyme activity is not too significant. Thus, immobilized lipase from
Aspergillus niger is feasible to synthesize biodiesel repeatedly. In second cycle and third cycle, there is an increase
of relative activity. This occurred because the catalytic side of enzyme in second cycle is not work effective to
convert the substrate, while in third cycle, all of catalytic sides is succeed converting substrate to biodiesel.

Relative Activity (%)

Cycle
Fig. 6. Stability of lipase from Aspergillus niger immobilized on resin.

4. Conclusion

Chitosan adsorption to resin is more effective in immobilization adsorption-crosslinking of lipase enzyme. The
highest enzyme loading, activity, and stability are reached by commercial lipase enzyme immobilized on anion-
exchange macroporous resin with add the chitosan on resin directly. This enzyme has unit activity of 24.69 U/g
support and yield of biodiesel is 50.79%. As biocatalyst, this enzyme is stable to use until fourth cycle with residual
activity as 63.94%. This optimum method is applied to lipase from Aspergillus niger. Lipase from Aspergillus niger
have unit activity of 22.84 U/g support and yield of biodiesel is 69.31%. This enzyme is also stable until fourth
usage with residual activity of 70.61%. Based on enzyme loading, activity, and stability of lipase from Aspergillus
niger is not too far from commercial lipase, so lipase from Aspergillus niger is feasible to use as biocatalyst in many
reaction.

Acknowledgements

The authors acknowledge support from Universitas Indonesia and Ministry of Research, Technology, and Higher
Education Republic of Indonesia in this research.

References

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[2] W. Tischer, and F. Wedekind. (1999) “Immobilized Enzymes: Methods and Application.” Springer Verlag Berlin Heidelberg 200 (1999)
[3] B. Brena, and F. Batista-Viera. Immobilization of Enzyme: A Literature Survey. Spain: Humana Press; 2006.
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