Microsoft Word - 164.docx


CHEMICAL ENGINEERING TRANSACTIONS 

VOL. 56, 2017 

A publication of 

The Italian Association 
of Chemical Engineering 
Online at www.aidic.it/cet 

Guest Editors: Jiří Jaromír Klemeš, Peng Yen Liew, Wai Shin Ho, Jeng Shiun Lim 
Copyright © 2017, AIDIC Servizi S.r.l., 

ISBN 978-88-95608-47-1; ISSN 2283-9216 

Production of Lipolytic Enzymes Using 
Agro-Industrial Residues 

Laura M. Pinotti*,a, Juliano X. Lacerdaa,  Marina M. Oliveiraa, Rogério D. Teixeiraa, 
Celson Rodriguesb, Sérvio T. A. Cassinib 
aDepartment of Engineering, Federal University of Espírito Santo, Rodovia BR 101 Norte Km 60 São Mateus – ES – Brazil 
bDepartment of Environmental Engineering, Federal University of Espírito Santo, 29075-910 Vitória – ES – Brazil  
 pinotti2008@hotmail.com 

Lipase (E.C.3.1.1.3) is a versatile and key enzyme in various bioprocesses involving the esterification and 
transesterification reactions for biodiesel generation. However, the production and recovery of enzymes is very 
expensive and often constitute an obstacle to wide use in bioprocesses. In this context, the solid state 
fermentation (SSF) could be an low-cost alternative, since it allows the use of agro-industrial wastes with low 
added value and promoting the production of more concentrated biocatalyst. The utilisation these residues in 
the fermentations, not only minimize the quantity off these residues in the environmental, but also add value to 
raw material, trough the production of economical interesting substances. The objective of this work is to verify 
the lipase production using pretreated sugarcane bagasse as substrate inoculated with lipolytic microorganism. 
The sugarcane bagasse is an agro industrial residue with high availability in Brazil and can have multiple uses, 
representing 25 % to 30 % of the total weight of the sugar cane. The microorganisms used were Penicillium sp. 
and Rhizomucor sp. combined with temperature operating conditions of 28 °C, 33 °C and 38 °C, moisture 
content of 60 %, 70 % and 80 % and olive oil concentration 5 %, 7.5 % and 10 % as inducer. The study was 
performed by using factorial design of type (33) with central points. The results shown that best microorganism 
for lipase production was the Rhizomucor sp. (0.58 IU/gsubstrate), although there is very slight difference when 
compared to the Penicillium sp. (0.47 IU/gsubstrate). These results were found in the conditions of 33 °C, 80 % 
moisture content and 10 % of inducer for both microorganisms. After analyses statistic was verified that the 
moisture content of the medium interfered in the enzyme production for Penicillium sp. For the Rhizomucor sp. 
the moisture content and the concentration of the inductor olive oil interfered in the enzyme production. 

1. Introduction

The use of enzymes as biocatalysts in industrial processes has been widely studied and it has shown promising 
for the production of high value-added compounds. According to Li et al. (2012), currently are known about 
4,000 enzymes and of these, around 200 are used commercially. Among the enzymes used as biocatalysts 
stand out lipases (E.C.3.1.1.3), which constitute the most important group of biocatalysts for biotechnological 
applications (Hasan et al. 2006). They catalyse the hydrolysis of fats and oils releasing fatty acids, diglycerides, 
monoglycerides and glycerol. These enzymes also catalyse esterification, transesterification and 
interesterification when the present amount of water is sufficiently low as to shift the thermodynamic equilibrium 
towards synthesis. These enzymes have a wide range of substrates, are stable to temperature changes and 
different pH and concentrations of organic solvents and also catalyse reactions showing a high enantioselectivity 
(Krieger et al., 2004). As lipases display a high degree of specificity in esterification and transesterification 
reactions, they became a principal biocatalyst for the production of several fine oleochemicals (Mustafa et al., 
2016).Lipases are commonly found in nature and can be obtained from animal sources (pancreatic lipase, 
gastric, and liver), plant and microbial (Damaso et al., 2008). Both eukaryotic microorganisms (yeast and fungi) 
and prokaryotic (bacteria, including actinomycetes) are lipase producers and their properties vary according to 
the origin. Microbial enzymes are most often used because of the wide variety of catalytic activity, ease of 

 

DOI: 10.3303/CET1756317

 
 

 
 

 
 
 
 
 
 
 

 
 
 
 
 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Please cite this article as: Pinotti L.M., Lacerda J.X., Oliveira M.M., Teixeira R.D., Rodrigues C., Cassini S.T.A., 2017, Production of lipolytic 
enzymes using  agro-industrial residues, Chemical Engineering Transactions, 56, 1897-1902  DOI:10.3303/CET1756317   

1897



genetic manipulation, rapid growth, are more stable, their production is more convenient and safer (Wiseman, 
1995). Microorganisms of the same strain have the potential to produce enzymes with features completely or 
partially differentiated, and therefore the search for new microbial sources remains focus of many researchers. 
A potential tool of interest in obtaining enzymes is in solid state fermentation (SSF), which allows the use of 
waste from other industrial processes as substrate and support for the growth of microorganisms, thus reducing 
the cost of production of enzymes (Silva et al., 2002). The SSF is based on the growth of microorganisms on a 
solid substrate with low water activity and has several advantages compared to the submerged fermentation 
such as space savings, simplicity in fermentation media, simple equipment and easy to control, high production 
yields , lower energy demand, and a higher concentrated metabolite of interest (Ângelo et al., 2014). Water 
absorption is critical to the success of this process and for this reason fibrous substrates are employed, and 
these provide the nutrients required for cell growth. Several agro-industrial waste can be used as support for 
the SSF. Mohseni et al. (2012) studied several agricultural products for lipase production, including rice bran, 
sugarcane bagasse, wheat bran, barley bran and corn meal. Amin et al.(2014) also realised a screening of 
different agricultural wastes such as rice bran, wheat bran, canola seed oil cake, sunflower hulls and peanut 
shells for the production of lipase by Aspergillus Melleus. Liu et al. (2014) used a mixture of sugarcane bagasse 
and sunflower seed cake as di-substrates for lipase production from Burkholderia cenocepacia, The utilisation 
these residues in the fermentations, not only minimise the quantity off these residues in the environmental, but 
also add value to raw material, through the production of economical interesting substances.  
Although SSF displays the benefits described above, there are some limitations such as the choice of 
microorganisms capable of growing under reduced moisture conditions, control and monitoring of parameters 
such as pH, temperature, humidity and air flow. Within this context, the objective of this study was to evaluate 
lipase production by solid state fermentation using Penicillium sp. and Rhizomucor sp., and sugarcane bagasse 
pretreated with acid-base solution as support and source of nutrients. We investigated the effect of temperature, 
moisture and concentration of olive oil as inducer, through the factorial design 3³.  

2. Materials and methods 

2.1 Microorganisms 
The Penicillium sp. and Rhizomucor sp. were isolated and provided by the research group of Department 
Environment Engineering of Federal University of Espírito Santo and kept agar slant (potato dextrose agar 
3.9 %) and frozen in glycerol solution 20 % (- 80 °C). 

2.2 Preparation of Inoculum 
The fungus was grown on agar plate containing PDA 3.9 % at 28 °C. The time required to obtain inoculum was 
studied and therefore samples were collected every 24 h and analysed spores concentration. The spores were 
scraped with 10 mL twen 80 (0.1 %v/v) and counted in a Neubauer chamber. 

2.3 Solid state fermentation 
For enzyme production was used sugarcane bagasse with particle diameter sizes between 0.6 mm and 2 mm 
and pretreated with acid-base solution. The sugarcane bagasse (natural and pretreated) was characterised 
according to Morais et al. (2010). This substrate was dried at 55 °C for 24 h. The bagasse was then added to 
Erlenmeyer flasks (10 g) and moistened with Mandel’s mineral salt solution to obtain the desired moisture. The 
flasks were autoclaved at 121 °C for 20 min and, after cooling, inoculated with 1 mL of liquid inoculum with a 
concentration of 108 spores/mL. We studied the cultivation temperature (28 °C, 33 °C and 38 °C), moisture 
content (60 %, 70 % and 80 %) and the concentration of inductor olive oil (5 %, 7.5 % and 10 %) for the time 
cultivation of 120 h (Table 1). For the experiments we used a factorial design 3³, with three central points, totaling 
29 experiments for each microorganism. Statistical analysis of the data were performed in Statistic v. 13.0 and 
the values were considered significant when p-value < 0.05. 

Table 1: Variables and levels used in the experiments 

 -1 0 +1 
Temperature of cultivation (°C) 28 33 38 
Moisture content (%) 60 70 80 
Concentration of inductor (%)   5      7.5 10 
 
The enzyme extraction was carried out using 100 mL of solution NaCl 2 %(m/v). The mixture of fermented solid 
(10 g) and the NaCl solution (100 mL) were placed in an orbital shaker for 1 h at 200 rpm and 29 °C. The mixture 
was first filtered and then centrifuged for 30 min at 6,000 g. The supernatant obtained was used for determination 
of enzyme activity. 

1898



2.4 Enzymatic activity 
The determination of the hydrolytic activity of the enzyme was performed measuring the hydrolysis of p-
nitrophenyl butirate (pNPB) in 2-propanol at 25 °C with the addition of 0.01 g of lipase. For this reaction, a 
volume of 29 mL of 100 mM phosphate buffer pH 8.0, was added to 1.0 mL of pNPB 15 mM in 2-propanol in a 
stirred and jacketed reactor and then the enzyme addition. The change in absorbance at a wavelength of 410 nm 
was monitored for 10.5 min. One unit of activity (IU/gsubstrate) was defined as the amount of enzyme required to 
produce one µmol of p-nitrophenol (pNP) per min. 

3. Results and discussion 

3.1 Kinects of spores production 
The spores production of Penicillium sp. and Rhizomucor sp. were studied at 28 °C, carrying out spores counting 
every 24 h. The results are shown in Figure 1 (Penicillium sp.) and Figure 2 (Rhizomucor sp.). 

 

Figure 1: Results of kinetic profile of spores production of Penicillium sp. 

 

Figure 2: Results of kinetic profile of spores production of Rhizomucor sp. 

It can be seen that the maximum production was approximately of 120 h, therefore we selected these time for 
carrying out the inoculation with the fungus. 

-2

0

2

4

6

8

10

12

14

0 50 100 150 200

C
o

n
ce

n
tr

a
ti

o
n

 o
f 

sp
o

re
s 

( 
1

0
8
/m

L)

Time (h)

0

0.5

1

1.5

2

2.5

3

3.5

4

0 50 100 150 200

C
o

n
ce

n
tr

a
ti

o
n

 o
f 

sp
o

re
s 

( 
1

0
8
/m

L)

Time (h)

1899



3.2 Characterisation of lignocellulosic biomass 
The results of characterisation of sugarcane bagasse (natural and pretreated with acid-base solution) are 
presented in Table 2.  

Table 2: Composition lignocellulosic of sugarcane bagasse 

Fraction Natural Sugarcane Bagasse Pretreated Sugarcane Bagasse 
Lignin (%) 22.8   8.1 
Cellulose (%) 29.7 62.6 
Hemicellulose A (%) 25.2 13.6 
Hemicellulose B (%) 13.5   9.6 
Total 91.2 93.9 
 
The pretreatment of sugarcane bagasse with the acid-base solution was effective in removal of lignin and 
hemicellulose, improving the availability of the cellulose for the microorganisms. 

3.3 Solid state fermentation (SFF) 
Results of enzyme production from the fungus are presented in Table 3. It can be seen that the lipase production 
was better in the conditions of 33 ºC, 80 % moisture content and 10 % of inducer for both microorganisms.  

Table 3: Results obtained in the fermentations using Penicillium sp. and Rhizomucor sp. 

Run T (°C) 
Moisture 

content (%) 
Concentration of 

inductor (%) 

Enzyme activity 
(IU/gsubstrate) 

Penicillium sp. 

Enzyme activity 
(IU/gsubstrate) 

Rhizomucor sp. 
1 28 60 5 0.142 0.219 
2 28 70 5 0.156 0.240 
3 28 80 5 0.211 0.333 
4 28 60 7.5 0.144 0.227 
5 28 70 7.5 0.167 0.260 
6 28 80 7.5 0.243 0.345 
7 28 60 10 0.189 0.289 
8 28 70 10 0.199 0.298 
9 28 80 10 0.301 0.412 
10 33 60 5 0.144 0.222 
11 33 70 5 0.166 0.257 
12 33 80 5 0.234 0.320 
13 33 60 7.5 0.168 0.238 
14 33 70 7.5 0.186 0.259 
15 33 80 7.5 0.366 0.502 
16 33 60 10 0.179 0.256 
17 33 70 10 0.234 0.295 
18 33 80 10 0.470 0.583 
19 38 60 5 0.154 0.224 
20 38 70 5 0.172 0.267 
21 38 80 5 0.342 0.335 
22 38 60 7.5 0.135 0.247 
23 38 70 7.5 0.187 0.301 
24 38 80 7.5 0.198 0.321 
25 38 60 10 0.135 0.239 
26 38 70 10 0.177 0.288 
27 38 80 10 0.187 0.335 
28 33 70 7.5 0.170 0.267 
29 33 70 7.5 0.188 0.271 

 
The experimental data were statistically analysed by analysis of variance (ANOVA) and the results are shown 
in Table 4. After statistical analysis it was found that the moisture (L) was the variables that interfered in the 
enzyme production (p < 0.05) when using Penicillium sp., obtaining the Eq(1). Higher moisture values led to 
better production. 

1900



IU gsubstrate⁄ =  0.204966 +  0.064556 M  (1) 

For fermentations with the Rhizomucor sp. it has been found that the moisture (L + Q) and the concentration of 
inductor (L) influenced the results of lipase production, resulting Eq(2). 

IU gsubstrate⁄ =  0.300148 +  0.073611 M −  0.020361 M
2 +  0.032111 I   (2) 

Table 4: The results of analysis of variance (ANOVA) for enzyme production using Penicillium sp. and 

Rhizomucor sp. 

 
Sum of 
Squares 

DF Mean square F-Value P-Value 

Penicillium sp.      
Temperature (ºC)(L) 0.000235 1 0.000235   0.08821 0.769246 
Temperature (ºC)(Q) 0.012543 1 0.012543   4.71406 0.040979 
Moisture Content (%)(L) 0.075014 1 0.075014 28.19179 0.000025 
Moisture Content (%)(Q) 0.010851 1 0.010851   4.07797 0.055793 
Concentration of Inductor (%)(L) 0.006806 1 0.006806   2.55768 0.124023 
Concentration of Inductor (%)(Q) 0.001576 1 0.001576   0.59247 0.449652 
Error 0.058538 22 0.002661   
Total SS 0.163253 28    
Rhizomucor sp.      
(1)Temperature (ºC)(L) 0.000242 1 0.000242   0.10110 0.753517 
Temperature (ºC)(Q) 0.007525 1 0.007525   3.14357 0.090074 
(2)Moisture content (%)(L) 0.097535 1 0.097535 40.74538 0.000002 
Moisture content (%)(Q) 0.012984 1 0.012984   5.42402 0.029445 
(3)Concentration of inductor (%)(L) 0.018560 1 0.018560   7.75358 0.010808 
Concentration of inductor (%)(Q) 0.000168 1 0.000168   0.07012 0.793633 
Error 0.052663 22 0.002394   
Total SS 0.187860 28    

4. Conclusions 

It can be concluded that both microorganisms produce lipase by solid state fermentation and the sugarcane 
bagasse is a good substrate. The moisture content is a variable that influenced the lipase production when was 
used the Rhizomucor sp. as well as Penicillium sp. being therefore a variable that requires further study. 

Acknowledgments 

The authors thank FAPES and UFES for the financial aid granted to carry out this work. 

References 

Amin M., Bhatti H.N., Zuber M., Bhatti I.A., Asgher M., 2014, Potencial use of agricultural waste for the 
production of lipase by Aspegillus melleus under solid state fermentation, Journal of Animal and Plant 
Science 24 (5), 1430-1437. 

Ângelo T., Da Silva R.R., Cabral H., 2014, Concomitant production of peptidases and lipases by fungus using 
agro-industrial residue in solid-state fermentation, International Journal of Current Microbiology and Applied 
Science 3 (5), 810-823. 

Damaso M.C.T., Passianoto M.A., De Freitas S. C., Freire D.M.G., Lago R.C.A., Couri S., 2008, Utilisation of 
agroindustrial residues for lipase production by solid-state fermentation. Brazillian Journal of Microbiology 
39, 676-681. 

Hasan F., Shah A.A., Hameed A., 2006, Industrial applications of microbial lipases, Enzyme and Microbial 
Technology 39, 235-251. 

Krieger N., Bhatnagar T., Baratti J.C., Baron A.M., Lima V.M.G., Mitchell D., 2004, Non- aqueous biocatalysis 
in heterogeneous solvent systems, Food Technology and Biotechnology 42 (4), 279–286. 

Li S., Yang X., Yang S., Zhu M., Wang X., 2012, Technology prospecting on enzymes: application, marketing 
and engineering, Computational and Structural Biotechnology Journal 2 (3), 1-11. 

1901



Liu Y, Li C., Wang S., Chen W.,2014, Solid-supported microorganism of Burkholderia cenocepacia cultured via 
solid state fermentation for biodiesel production: Optimisation and kinetics, Applied Energy 113, 713–721. 

Mohseni S., Najafpour G.D., Vasegui Z., Mahjoub S., 2012, Solid state fermentation of agricultural residues for 
lipase production in a tray-bioreactor, World Applied Sciences Journal 6 (7), 1034-1039. 

Morais J.P.S, Rosa M. F., Marconcini J.M. 2010. Procedures for lignocellulosic analysis (in Portuguese). 
EMBRAPA, Campina Grande, PB. 

Mustafa A., Karmali A., Abdelmoez, W. 2016. Optimisation and economic assessment of lipase-catalysed 
production of monoesters using Rhizomucor miehei lipase in a solvent-free system. Journal of Cleaner 
Production 137, 953-964 

Silva D., Martins E.S., Silva R., Gomes E., 2002, Pectinase production by Penicillium viridicatum RFC3 by solid 
state fermentation using agricultural wastes and agro-industrial by-products, Brazillian Journal of  
Microbiology 33, 318-324. 

Wiseman A., 1995, Introduction to principles. In: Wiseman, A (Ed.) Handbook of enzyme biotechnology, 3rd 
edition, Padstow, Cornwall, Ellis Horwood Ltd., T.J Press Ltd, UK, 3 -8. 

1902