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Energy Procedia 57 (2014) 860 – 866

2013 ISES Solar World Congress

Evaluation of agro-industrial wastes to produce bioethanol:

case study - mango (Mangifera indica L.)
Santis Espinosa L. Fernandoa*, Peréz-Sariñana Bianca Ya., Saldaña-Trinidad
Sergiob, D. Eapenc, P.J. Sebastiana*
a
Instituto de Energías Renovables UNAM, Privada Xochicalco s/n CP 62580 Temixco, Morelos, México.
b
Universidad Politécnica de Chiapas, calle Eduardo J. Selvas s/n Col. Magisterial. CP 29010 Tuxtla Gutiérrez, Chiapas, México.
c
Instituto de Biotecnología-UNAM, Av. Universidad 2001, Cuernavaca, Morelos, 62210, México.

Abstract

This study was carried out to evaluate the fermentation performance and behavior of the yeast
Saccharomyces cerevisiae Y2034 in mango pulp and peel (Mangifera indica L.). In this paper we studied the
fermentation of mango (pulp and peel) a fruit rich in Mexico with high sugar content. The process for producing
ethanol comprised the following steps: substrate preparation, preparation of the YPD medium, growth of the yeast S.
cerevisiae Y2034, alcohol fermentation and distillation. In this study we utilized the raw materials amounting 75
percent pulp and 25 percent peels and seeds. The maximum growth of yeast was observed in the treatment of
pulp and peel with 150 g/L of initial reducing sugar, obtaining 2.6×108 cfu mL−1. All the experiments were
carried out in triplicate.

© 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
© 2013 The Authors. Published by Elsevier Ltd.
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and/or
Selection and/or peer-review
peer-review under responsibility
under responsibility of ISES. of ISES

Keywords: S. cerevisiae Y2034; Mango (Mangifera indica L.); fermentation; bioethanol.

* Corresponding authors. Tel.: +52 961 190 9340, +52 777 3620090
E-mail address: lfsae@ier.unam.mx, sjp@ier.unam.mx

1876-6102 © 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Selection and/or peer-review under responsibility of ISES.
doi:10.1016/j.egypro.2014.10.295
 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866 861

1. Introduction

Currently bioethanol is one of the most studied biofuel worldwide, because it can be a sustainable
alternative for replacing fossil fuel [1]. In the case of Mexico, there has been boosting production in
response to the shortage of fossil fuels nationally and internationally. Sugar cane has been one of the most
used raw materials in the production of bioethanol. Brazil and USA are the world’s largest bioethanol
producers, using sugarcane and corn as the respective feedstocks. In the sugarcane industry, large
amounts of lignocellulosic materials (sugarcane bagasse) are produced during sugar and ethanol
production. Sugarcane bagasse is currently used as a fuel for supplying the energy required for the plant,
while sugarcane trash, in earlier days was burnt to improve the cultivation, is today mostly left in the field
for agricultural purposes [2]. However, there are various raw materials and wastes with a high content of
fermentable sugars that can be evaluated for bioethanol production.

Mango (Mangifera indica L.) may be a case for this purpose because it is one of the most popular and
abundant tropical fruits in Southeast Asia and is often in oversupply after each harvest [3]. This fruit is
mainly grown in 85 countries. Asia and Eastern countries produce about 80 percent of total world
production. There are a great number of varieties, which differ from each other with respect to crop, the
color of the skin, pulp, the flavor and aroma of the fruit, among other features [4].

Mexico is the fifth producer of mango around the world. The main producers are India, China, Thailand,
Pakistan and Mexico, followed by Indonesia. Together, these six nations generate three of every four tons
of the fruit produced worldwide. The area of cultivation of mango in Mexico amounts to 186,505 hectors.
Mexico has a production of 1.481 million tons according to the data reported by the SIAP in 2012 [5].
On the other hand, according to the latest report of SAGARPA, there is a history of losses amounting to
230 thousand tons per year. According to a report published by Reddy and Reddy in 2005, Mango
contains a high concentration of sugar (16–18% w/v) and acids with organoleptic properties, and also
contains antioxidants. Sucrose, glucose and fructose are the principal sugars in ripe mango, with small
amounts of cellulose, hemicellulose and pectin [6].

2. Materials and methods

2.1. Raw material

The raw material (Mango Ataulfo (Mangifera indica L.)), which was employed for this work was dried
with a solar dehydrator and milled. After the pretreatment, we conducted a thermal hydrolysis process
(THP) at conditions of 130 °C and 15 psi for 20 min. After the pretreatment, it was centrifuged at 5000
rpm and 28 °C for 10 minutes (Centrifuge 5804R, 15 amp version) to clarify the substrate. The substrate
was adjusted to a concentrations of reducing sugar of 100, 150 and 200 g/L, respectively according to the
proposed experimental design.

2.2. Microorganism and inoculum preparation

862 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866

The strain Saccharomyces cerevisiae Y2034 was provided by the ceparium of the Polytechnic University
of Chiapas. Yeasts were maintained in sterile YPD solid culture media consisting of yeast extract,
peptone, glucose and agar at concentrations of 10, 20, 20 and 18 g/L, respectively. They were stored at 4
°C. To prepare the inoculums of S. cerevisiae Y2034 a loop-full of cells were added to each 150 mL tube
containing 100 mL sterile (121°, 20 min, 15 psi.) YPD medium liquid consisting of glucose, peptone and
yeast extract at concentrations of 20, 20 and 10 g/L, respectively. The test tubes were incubated in a
rotary shaker at 30 °C and 150 rpm for 24 h. The preparation protocol was in accordance with the
literature [7] [8].

2.3 Fermentation

At the end of incubation, the contents of these tubes were used for fermentation. Fermentations were also
carried out in 150 mL Erlenmeyer flasks with 100 mL medium, for 24 h. The agitation was fixed at 150
rpm (Temperature controlled shaker, Lusmistell models iro 70), at a pH of 4.5 and at 30 °C. The flasks
were inoculated with 1x106 CFU/mL of S. cerevisiae Y2034. An Erlenmeyer flask containing hydrolysate
with the addition of 5 g/L yeast extract, 5 g/L peptone, 5 g/L KH 2PO4, 0.2 g/L (NH4)2SO4 and 0.4 g/L
MgSO4.7H2O was used. All experiments were carried out in triplicate. Samples were taken at 0 h, 6 h, 12
h, 18 h and 24 h to analyze consumption of sugar and growth of yeast [7] [9].

2.4 Experimental design

The experimental strategy for the alcohol fermentation of the mango was carried out using the software,
design-expert 8.0.7.1 (version 8.0.3, stat-ease, inc., minneapolis, USA). It is based on the methodology of
response surfaces (RSM) based on the central composite design (CCD) 2 2 factorial.

2.5. Analytical methods and calculations

The total soluble solids (°Brix) and pH were measured at the indicated time points by using a pocket
digital refractometer (SPER SCIENTIFIC, MOD. 300053) and a pH meter (OAKTON), respectively [10].
Appropriate dilutions from the growing culture suspensions were made. The cellular viability was
determined by the Methyl Blue staining technique of direct counting using the neubauer camera [11]. The
sugar concentration was estimated by the DNS technique or 3,5 Dinitrosalicílic acid method [12]. Each
sample was analyzed in triplicate.

3. Results and discussion

3.1. Experimental strategy of alcohol fermentation

For mango fermentations the concentration of reducing sugar and yeast concentration were taken as input
variables. Therefore, based on the proposed design we obtained the behavior of sugar consumption and
cell density as shown in table 1. One can see that the treatments with 150 g/L initial reducing sugar using
the pulp and peel show higher cell growth and sugar was reduced to 89 percent.
 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866 863

Table 1. Experimental design for the alcohol fermentation of mango.

Input Variables Output Variables

Sugar Sugar
concentration Cell Concentration (UFC/mL) consumption
(g/L) (g/L)
Initial Initial A B A B
1 100 1.00E+06 3.00E+07 5.00E+07 32.99 15.33
2 100 1.00E+06 3.50E+07 5.00E+07 32.1 14.4
3 100 1.00E+06 3.50E+07 6.50E+07 33.8 15.2
4 150 1.00E+06 8.70E+07 2.50E+08 47.11 17.94
5 150 1.00E+06 7.50E+07 2.55E+08 46.8 17.5
6 150 1.00E+06 9.50E+07 2.60E+08 47.33 16. 70
7 200 1.00E+06 1.00E+08 8.00E+07 80.1 47.25
8 200 1.00E+06 9.00E+07 9.00E+07 80.1 46.5
9 200 1.00E+06 1.05E+08 9.50E+07 81.01 46.85
A = pulp
B = pulp + peel

3.2. Fermentations with mango pulp

In the process of alcohol fermentation, the kinetic data were obtained for the sugar and yeast. The
behavior for each treatment is shown in Figure 1. These fermentations were carried out only with mango
pulp. The results of these treatments showed that higher cell densities are given with 200 g/L of initial
sugar, however reducing sugar in the sample concentration is not recommended for alcohol fermentation
of mango pulp at high concentrations, given the fact the sugar is not completely metabolized and
therefore there is a loss of 40 percent of the reducing sugar.
864 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866

Fig. 1. Kinetic behavior of yeast and reducing sugar during alcohol fermentation with initial concentrations of 200 (♦), 150 (*) and
100 (●), g/L respectively of mango pulp.

Figure 2 shows the behavior of total soluble solids (SST), temperature and pH. It is noted that for the first
parameter, decreasing ° Brix are higher than 50 percent of initial values. However, as demonstrated in
figure 1 treatment with 200 g/L of initial sugar had the highest cell density. But, the best treatment is 150
g/L, because the sugar is reduced up to 70 percent and the cell density is very close to the higher density
treatment. The temperature was stable at 30 ° C, as the pH is maintained in the range reported for the
optimal development of this yeast (4.5-5).

Fig. 2. Behavior of the temperature (◊), pH (□) and the SST with initial concentrations of 13.7 (∆), 17 (○) and 22.3 (*),°Brix
respectively using mango pulp.

3.3. Fermentations with pulp and mango peel

During the development we proposed different experimental treatments, first with the sugar obtained
directly from mango pulp. Later, treatments have been proposed using the full mango and using cellulosic
waste as shown in Table 1. These fermentations were performed with pretreatments for obtaining most
amount of reducing sugar. Figure 3 shows the results obtained from fermentation of the complete mango
with different initial sugar. In this graph, it can be seen that the best treatment is the initial concentration
of 150 g/L, because it is possible to obtain the highest number of cells and increased metabolization of
sugar very close to 90 percent.
 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866 865

Fig. 3. Kinetic behavior of yeast and reducing sugar during alcohol fermentation with initial concentrations of 200 (♦), 150 (*) and
100 (●), g/L respectively using mango pulp and peel.

In these treatments the behavior of total soluble solids (° Brix), temperature and pH can be seen in figure
4, where it is displayed the treatments with 200 and 100 g/L initial sugar respectively. It is seen that there
is a decrease of more than 50 percent of ºBrix compared to the initial values. However, as demonstrated in
figure 3, treatment with cell density was increased to 150 g/L of initial sugar and hence is the best
treatment. In this treatment the sugar was reduced to around 90 percent. At the same time, the temperature
was stable at 30 °C and the pH was maintained in the range reported for the optimal development of this
yeast (4.5-5).

Fig. 4. Behavior of temperature (◊), pH (□) and the SST with initial concentrations of 13.7 (∆), 17 (○) and 22.3 (*), °Brix
respectively using mango pulp and peel.

4. Conclusion

Based on the results obtained in this study, the best concentration of initial sugar for fermentation of
mango is 150 g/L. Under these conditions the cell number and sugar consumption were better than the
other experimental treatments. The pulp, peel and seed of the mango provide fermentable sugars for the
production of bioethanol. We established an appropriate methodology for alcohol fermentation of
agricultural waste from mango for the evaluation of the production of bioethanol. The observed changes
in the kinetics of the yeast are related to the composition of the medium, since the temperature and pH are
866 Santis Espinosa L. Fernando et al. / Energy Procedia 57 (2014) 860 – 866

stable.

Acknowledgements
The authors thank the technical help received from M.C. Jose Campos in spectrophotometry analysis, Dr.
Patricia Altuzar in FTIR measurement and M.C. Gildardo Casarubias in the technical help in general.

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