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Fermentation of pineapple fruit peel wastes for bioethanol production

Article  in  Waste and Biomass Valorization · May 2019

DOI: 10.1007/s13399-019-00436-y

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Biomass Conversion and Biorefinery (2019) 9:761–765
https://doi.org/10.1007/s13399-019-00436-y

ORIGINAL ARTICLE

Fermentation of pineapple fruit peel wastes

for bioethanol production
Jennifer T. Casabar 1 & Yuwalee Unpaprom 2 & Rameshprabu Ramaraj 1

Received: 14 April 2019 / Revised: 6 May 2019 / Accepted: 8 May 2019 / Published online: 21 May 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
In the past few years, the global demand in bioethanol has been continuously rising due to its economic importance. Several
countries have exerted considerable efforts in the production of bioethanol as a transport fuel from different feedstocks and started
to practice consuming this environment-friendly fuel. Thailand is now one of the top consumers of bioethanol in South East Asia
and belongs to the top 5 leading countries in producing pineapples. Production of bioethanol from pineapple wastes, especially
the fruit peels, became possible. In this study, the effects of alkaline pretreatment and microbial hydrolysis through Trichoderma
harzianum of pineapple fruit peel were evaluated. Among the four concentrations of NaOH, the 0% NaOH gave the highest total
and reducing sugar (458.44 ± 13.6 g/L and 279.67 ± 21 g/L) than 1%, 3%, and 5% concentrations of NaOH. Samples pretreated
with 0% NaOH were subjected to microbial hydrolysis which showed an increase in reducing sugar of the samples. At the end of
the experiment, a bioethanol yield of 5.98 ± 1.01 g/L from pineapple fruit peel was successfully produced at 48 h of fermentation.

Keywords Bioethanol . Pineapple fruit peel . Trichoderma harzianum . Alkaline pretreatment

1 Introduction
Pineapple (Ananas comosus) is a popular tropical fruit having
a short stem and slender hard leaves that grow to medium- to
large-sized fruit. In 2017, the pineapple was reported to be the
second most important tropical fruit in the world production
having an estimated global production of 25.9 million tons
[1]. These large productions of pineapple were headed by
the top 5 leading countries, namely, Costa Rica which pro-
duced 3056.45 metric tons of pineapples annually and follow-
ed by the Philippines (2671.71 metric tons), Brazil (2253.9
metric tons), Thailand (2123.18 metric tons), and India
(1861 metric tons) [2]. This edible, nutritious fruit can be
consumed fresh, dried, or processed, and its leaves and crowns
can also be used as a feed supplement for livestock. Aside
from these, pineapples are now being used by several
manufacturing companies of medicines and cosmetics due to
* Rameshprabu Ramaraj
rrameshprabu@gmail.com; rameshprabu@mju.ac.th
1
School of Renewable Energy, Maejo University, Sansai, Chiang
Mai 50290, Thailand
2
Program in Biotechnology, Faculty of Science, Maejo University,
Sansai, Chiang Mai 50290, Thailand
its useful compounds such as citric acid, bromelain, and anti-
inflammatory properties. People may also produce different
products like fabrics, papers, bags, and ropes through the fi-
bers of its leaves [3].
Since the global demand and consumption of pineapples
are intermittently rising, production of pineapple wastes is
also on its rise. Choonut et al. [4] stated that 0.62 million tons
of pineapple wastes is produced annually and continuously
increasing as the global production rose in recent years.
Proper waste disposal of pineapple’s organic residues (which
include the fruit peel and crown) is necessary especially for
the industrial companies that use pineapple as the main ingre-
dient in their products. However, the use of these wastes has
become an interest in the production of biofuels, mainly for
bioethanol production.
One of the most promising biofuels nowadays is the
bioethanol. Bioethanol has been known due to its various
advantageous applications in industry, transportation, and en-
ergy sectors. This chemical compound can be produced from
organic matters which contain a relative amount of carbohy-
drates such as corn, cassava, sugar beets, and other plants that
have high sugar content [5–8]. Since the 1980s, the production
of eco-friendly bioethanol is being done by several countries
such as the USA, Brazil, Ukraine, Pakistan, South Africa, and
other members of the European Union, and some countries in
762
Asia [9, 10]. These countries produce bioethanol as a transport
fuel that produces a reduced amount of greenhouse gases
when compared with conventional gasoline [10].
In recent studies, the production of bioethanol from
lignocellulosic was investigated to avoid the conflict of
edible materials for human consumption and industrial
purposes [11]. Aside from this, lignocellulosics from ag-
ricultural wastes, weeds, and micro- and macroalgal bio-
mass were used for value addition. One of these is the
study of Yücel and Göycıncık [12] in which the spent
tea waste gave 1.61% v/v (12.72 g/L) ethanol yield using
simultaneous saccharification and fermentation. Another
bioethanol production from the residual banana bulb was
also recorded to produce 310 kg/ton ethanol under the
same technique [13]. Since pineapple wastes such as its
fruit peel and crown are comprised of lignin, hemicellu-
lose, and cellulose, these are considered to be lignocellu-
losic materials that can be used in the production of
bioethanol. It has high fiber content which made it as a
potential bioethanol feedstock. Table 1 shows the chemi-
cal composition of pineapple wastes.
According to the study of Itelima et al. [15], the highest
optimal ethanol yield of 8.34% (v/v) of pineapple peel was
recorded using simultaneous saccharification and fermenta-
tion which was compared with banana and plantain. Another
study of bioethanol production from pineapple peel was done
by Choonut et al. [4] in which 1.23% v/v (which is also equal
to 9.69 g/L) of ethanol yield was produced after 72 h of fer-
mentation of hot water bath pretreated pineapple peel. The
latest study was conducted by Gil and Maupoey [16] regard-
ing pineapple waste valorization in which it was found out that
5.4 ± 0.1% v/v ethanol production was observed under simul-
taneous saccharification and fermentation. Several related
studies about bioethanol production from pineapple wastes
were conducted in different techniques and processes.
However, the use of NaOH for alkali pretreatment and the
application of microbial hydrolysis is not yet been done for the
production of bioethanol from pineapple fruit wastes. In this
study, the effect of alkali pretreatment and Trichoderma
harzianum in the hydrolysis of pineapple fruit peel for pro-
duction of bioethanol was evaluated.
Table 1 The chemical composition of pineapple wastes [14]
Composition Component (% dry matter)
Whole Skin Crown
Cellulose 19.4 14.0 29.6
Hemicellulose 22.4 20.2 23.2
Lignin 4.7 1.5 4.5
Ash 0.7 0.6 0.4
Cell soluble matter (CSM) 53.4 64.8 42.5
Biomass Conv. Bioref. (2019 ) 9:761–765
2 Materials and methods
2.1 Raw material and microorganisms
Pineapple fruit peel was collected from the local fruit vendors
from Sansai District, Chiang Mai, Thailand. The collected
pineapple wastes were manually cleaned by removing unnec-
essary debris and were cut into small pieces for faster drying
under the solar dryer. Afterward, dried samples were
powderized through milling and served as a working sample
for t he experiment. An alcohol-act ive dry yeast
Saccharomyces cerevisiae (Angel Yeast Co., Ltd., P. R.
China) and T. harzianum were purchased at Institute of
Product Quality and Standards, Maejo University, and were
used for fermentation and hydrolysis processes.
2.2 Alkali pretreatment
For the alkali pretreatment, methods used by Chaudhary et al.
[17] were followed with some modifications. Sodium hydrox-
ide at concentrations of 1%, 3%, and 5% was used to treat the
pineapple fruit peel at biomass loading of 5% (w/v). Zero per-
cent of NaOH was also employed in the samples to serve as a
control in the study. Treatment temperature of 30 °C was main-
tained for 48 h. This pretreatment was carried out in a 250-mL
Erlenmeyer flask in steady condition inside the incubator hav-
ing 30 °C maintained temperature. After alkali pretreatment, pH
was adjusted with 1 N HCl until it reached to the desired pH
depending on the designated hydrolysis condition. Pretreated
samples were analyzed for total sugar (TS) and reducing sugar
(RS) determination [18]. All samples were done in triplicate.
2.3 Hydrolysis
Saccharification and fermentation procedures were accom-
plished via methods used by Sindhu et al. [18] with the replace-
ment of the enzyme used in the study. Two percent of
T. harzianum inoculum was added to the best pretreatment con-
dition which was used as a source of enzyme instead of com-
mercial cellulase that was applied in the study. The pretreated
pineapple peel was hydrolyzed in 250-mL Erlenmeyer flasks
with 0.125% v/v surfactant (Tween 20) concentration. The sam-
ples were incubated at 30 °C, 200 rpm with a pH of 6 for 48 h.
These were centrifuged to remove the unhydrolyzed residue
after saccharification. The supernatant (hydrolysate) was used
for RS and TS analyses by the 2,5-dinitrosalicylic acid method
Pulp
[19] and phenol–sulfuric method [20] and was compared with
14.3 the sugars produced by unhydrolyzed samples.
22.1
2.3 2.4 Ethanol fermentation
0.2
61.4 Ethanol fermentation was carried out using the hydrolysate
generated from the best hydrolysis treatment based on the
Biomass Conv. Bioref. (2019 ) 9:761–765
number of sugars produced. The hydrolysates obtained after
microbial saccharification were filtered and centrifuged (4 °C,
1000 rpm, 15 min) to remove the unhydrolyzed residue.
Fermentation was done in a 250-mL Erlenmeyer flask with
175 mL hydrolysate at a pH of 5.6. This was inoculated with
2% S. cerevisiae (Angel Yeast Co., Ltd., P. R. China) and was
incubated at 36 °C for 72 h. The ethanol concentration deter-
mination was done every after 24 h of fermentation using
Ebulliometer (Dujardin-Salleron, Alcohol Burner, France).
3 Results and discussion
3.1 Effect of NaOH pretreatment in pineapple peel
A pretreatment method is essential in the efficient production
of bioethanol by enhancing the accessibility of enzymes to
cellulose of specific feedstock [21]. The use of alkaline pre-
treatment primarily deals with the removal of lignin of a plant
which protects and encloses other components of a plant such
as a hemicellulose and cellulose [22]. Since studies regarding
the use of alkali pretreatment in pineapple peel are limited, the
application of three concentrations of NaOH (1%, 3%, and
5%) was made to evaluate the effect of NaOH in the produc-
tion of sugars in pineapple peel. Figure 1 reveals the TS and
RS productions of pineapple peel after the pretreatment pro-
cess. NaOH pretreatment was carried out with 5% w/v bio-
mass loading. The results showed that the samples pretreated
with the control treatment (dH2O) gave the highest amount of
both TS (458.44 ± 13.6 g/L) and RS (279.67 ± 21 g/L) after
2 days of the pretreatment process.
The outcome also revealed that there was a decreasing
production of sugars as the NaOH concentration increases,
in which it ranged from 328.40 to 408.23 g/L of TS and 30
to 138.10 g/L of RS. This means that the relationship between
the NaOH concentration and sugar production in pineapple
peel is inversely proportional. According to Hajar et al. [23],
the pineapple fruit peel is initially acidic with the pH ranging
from 3.47 to 3.85, and pineapple peel was previously de-
scribed to have a lignin content of 1.5% of dry matter [14].
Sugar production after pretreatment
Sugar Concentration (g/L)
500
400
300
200
100
0
dH20 1% 3%
(Control)
NaOH Concentration
Total Sugar Reducing Sugar
Fig. 1 Sugar concentration of pineapple peel after pretreatment
763
It is a relatively small amount of lignin to be degraded by the
base chemical and a substantial portion of the NaOH applied
in the process reacted with the natural acid content of the
sample. Usually, when an acid and a base combined, it forms
salts and other inhibitory compounds (such as furans, phenol,
and carboxylic acid) which can affect the sugar and ethanol
production efficiency of the material [24]. Aside from this, the
crystallinity of the cellulose also affects the efficient produc-
tion of sugars from the feedstocks. Rahnama et al. [25] re-
vealed in her study that the alkali-pretreated rice straw showed
a 62.42% crystallinity of cellulose and 50.81% cellulose crys-
tallinity in untreated one. This coincides with the result of the
present study in which the resulting sugar concentrations were
higher in the control group than those samples pretreated with
1%, 3%, and 5% NaOH concentrations.
3.2 Influence of T. harzianum as microbial hydrolysis
of pineapple fruit peel
Based on the pretreatment result, pretreated samples with dis-
tilled water (control) gave the highest amount of total and
reducing sugar among the three concentrations of NaOH. In
this case, samples under 0% NaOH were used as a substrate to
undergo the hydrolysis process. Table 2 details the resulting
total and reducing sugar, as well as the degree of polymeriza-
tion after the hydrolysis.
As the results showed, the samples in the control group
produced a higher amount of total sugar than those of the sam-
ples hydrolyzed with 2% T. harzianum. However, the samples
hydrolyzed with T. harzianum were found to have a more sig-
nificant amount of reducing sugar and exhibit a lower degree of
polymerization than the control group. The higher reducing
sugar observed can be explained by the production of enzymes
by T. harzianum that is responsible for the degradation of hemi-
celluloses and celluloses present in the feedstock. It is also
important to calculate the degree of polymerization in order to
determine the number of monosaccharides formed in a macro-
molecule. A higher degree of polymerization requires intensive
degradation of structural units to extract the simple sugars like
glucose in the sample exhaustively. In the recent study, a lower
degree of polymerization under 2% T. harzianum after hydro-
lysis proved that T. harzianum could efficiently disrupt the
crystallinity of cellulose and hemicellulose to produce a higher
amount of fermentable sugars (see Table 2).
Cellulase production of T. harzianum was reported by sev-
eral studies which can reduce and degrade the crystallinity of
the cellulose of specific material [25–28]. In the study of
Rahnama et al. [25], T. harzianum was also observed to have
5%
carboxymethyl cellulase (CMCase) and β-glucosidase activi-
ty at 111.3 U/g substrate and 173.71 U/g substrate in untreated
rice straw. Aside from this, Delabona et al. [26] stated that the
combination of pectinase and α-L-arabinofuranosidase with
the enzymatic extract from T. harzianum may result to the
764
Table 2 Resulting sugar
concentrations after hydrolysis Hydrolysis treatment
with a degree of polymerization
Unhydrolyzed (control)
With 2% T. harzianum
hydrolysis efficiency of 116% and can be used for different
agricultural residues [29]. Since the application of
T. harzianum can produce higher fermentable sugar and gen-
erated a lower degree of polymerization in the present study,
this means that T. harzianum can positively affect the efficient
production of fermentable sugar from pineapple fruit peel.
3.3 Bioethanol production
In bioethanol production, anaerobic respiration is usually done
after the hydrolysis process. The resulting hydrolysates from
2% T. harzianum were used to proceed fermentation tech-
nique. It was successfully done by inoculating 2% v/v S.
cerevisiae (dry yeast) at the start of fermentation and incubat-
ed for 72 h at 36 °C. Samples were collected every after 24 h
to check the ethanol production. Figure 2 shows the ethanol
produced from the optimum condition for pineapple fruit peel.
As Fig. 2 demonstrated, the highest bioethanol production
was observed at 48 h of incubation having 5.98 ± 1.01 g/L,
followed by 24 h and 72 h of fermentation with 5.31 ± 1.76 g/
L and 4.5 ± 0.72 g/L of ethanol concentration, respectively.
The same trend was also observed in degree of polymerization
in which the highest DP was recorded in 24 h of fermentation.
From the results gathered, the 2% dry yeast was further acti-
vated with the sugars present in the sample after 24 h of fer-
mentation and resulted in 82.42% conversion ratio of ferment-
able sugars to ethanol from the start of fermentation up to 48 h
of incubation (refer to Tables 2 and 3). This result exceeds the
previously reported reducing sugar utilization ratio of 56.57%
of either S. cerevisiae or Pachysolen tannophilus in biologi-
cally pretreated sorghum husk [30].
Moreover, the amount of reducing sugar recorded within
the fermentation period correlates the trend in bioethanol
Bioethanol production
8 of fermentable sugars in pineapple fruit peels.
Ethanol concentration (g/L)
7
6
5 Table 3 Sugar concentrations of pineapple fruit peel within 72 h of
4 fermentation
3
2 Responses
1
0 24 h
24 hr 48 hr 72 hr
Fermentation time
Fig. 2 Bioethanol production of pineapple fruit peel from 72 h
fermentation
Biomass Conv. Bioref. (2019 ) 9:761–765
Total sugar (g/L) Reducing sugar (g/L) Degree of polymerization
458.44 ± 13.6 279.05 ± 20.67 1.64
394.52 ± 16.13 285.67 ± 17.21 1.38
production of pineapple fruit peels (see Table 3). As the re-
ducing sugar decreases, the ethanol production increases. This
is due to the utilization of fermentable sugars by S. cerevisiae
to convert it into alcohol. Nevertheless, it can be noticed that
there was a remaining amount of reducing sugars of 51.7 ±
8.78 g/L that was present at the end of fermentation. This is
because of the extra action of hydrolytic enzymes still present
in the samples [16]. In the study of Tropea et al. [31], the
highest ethanol production of 3.9% (v/v) or 30.77 g/L ethanol
from pineapple wastes was obtained after 30 h of the same
fermentation used in this study. Moreover, 5.4 ± 0.1% (42.61
± 0.79 g/L) of ethanol yield (highest) was achieved by Gil and
Maupoey [16] using SSF at 48 h of fermentation of pineapple
wastes which supported the results of the present study.
4 Conclusion
As a low-cost feedstock for ethanol production, pineapple
fruit peel successfully produced bioethanol using 2%
S. cerevisiae (dry yeast). In the present study, the use of
NaOH in the pretreatment of pineapple fruit peel was
found not to affect the high production of sugars.
Formation of salts and other inhibitory compounds is taken
into consideration when the NaOH and the natural acid
content of the sample react. Finally, T. harzianum for hy-
drolysis process positively enhances the production of re-
ducing sugars in 0% NaOH than the unhydrolyzed one.
Based on the present results, alkali pretreatment in pineap-
ple fruit peel is not suitable for efficient production of
sugar from this kind of feedstock, and T. harzianum inoc-
ulum can effectively increase the sugar content of the sam-
ple after hydrolysis. However, it is highly recommended to
use extracted and purified carbohydrate-binding module
(CBM) from T. harzianum for more efficient production
Fermentation time
48 h 72 h
Total sugar (g/L) 114.66 ± 2.9 172.00 ± 8.58 84.83 ± 6.99
Reducing sugar (g/L) 55.80 ± 8.2 50.22 ± 6.54 51.7 ± 8.78
Degree of polymerization 2.05 3.44 1.64
Biomass Conv. Bioref. (2019 ) 9:761–765
Acknowledgments Authors would like to thank School of Renewable
Energy, Program in Biotechnology, Faculty of Science, Maejo University,
Chiang Mai, Thailand, for providing the research facilities during the conduct
of the study.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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