Fuel 149 (2015) 85–89

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Fuel
journal homepage: www.elsevier.com/locate/fuel

Production of Bioethanol from agro-industrial wastes

Alma Rosa Domínguez-Bocanegra a,⇑, Jorge Antonio Torres-Muñoz b, Ricardo Aguilar López a
a
Departamento de Biotecnología y Bioingeniería, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Cd, de México, 07360 México, Mexico
b
Departamento de Control Automático, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Cd, de México, 07360 México, Mexico

h i g h l i g h t s

 Bioethanol production in coconut milk, pineapple juice and tuna juice was validated.
 Maximal ethanol production of 22% v/v in coconut milk.
 Pineapple juice fermentation reached the maximal ethanol productivity 0.47 g/g.

a r t i c l e i n f o a b s t r a c t

Article history: A suitable alternative to replace fossil fuels is the production of bioethanol from agro-industrial wastes.
Received 31 March 2014 Coconut, pineapple and tuna are fruits that available almost along the year in Mexico but a high percent-
Received in revised form 7 September 2014 age of these fruits are wasted by producers. The aim of this study was to investigate the using of agro-
Accepted 19 September 2014
industrial wastes natural carbon sources such as those present in coconut milk, pineapple juice and tuna
Available online 1 October 2014
juice, to promote the synthesis of bioethanol by yeast Saccharomyces cerevisiae CDBB 790. Cultures were
grown in 500 mL Erlenmeyer flasks containing 350 mL of culture media (YM medium, coconut milk, pine-
Keywords:
apple juice or tuna juice) the yeast cells were inoculated with 35 mL YM medium in the exponential
Bioethanol
Agro-industrial wastes
growth phase. Results show that the highest ethanol concentration obtained from was 20% v/v in coconut
Saccharomyces cerevisiae milk, 22% v/v in pineapple juice and 12% v/v in tuna juice. The consumption of sugars at 36 h was 88.62%
in coconut milk, 93.75% in tuna juice, 90.62% in pineapple juice and 98.6% in YM medium.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction agriculture from an environmental point of view so that the use

Bioethanol is an increasingly important alternative fuel for the
replacement of gasoline, with a world production in 2009 of
19,535 millions of gallons and an estimate, only for USA in 2022,
of 36,000 millions of gallons. It is thus expected that the produc-
tion of bioethanol will keep on increasing in the next 10 years
[1]. The ethanol obtained from biomass-based waste materials or
renewable sources is called as bioethanol and can be used as a fuel,
chemical feedstock, and a solvent in various industries. It has cer-
tain advantages as petroleum substitutes, viz., alcohol can be pro-
duced from a number of renewable resources, alcohol as fuel burns
cleaner than petroleum which is environmentally more acceptable.
It is biodegradable and thus, checks pollution. It is far less toxic
than fossil fuels. It can easily be integrated to the existing transport
fuel system, i.e., up to 5% bioethanol can be blended with conven-
tional fuel without the need for modification [2,3]. Agricultural
organic wastes are currently one of the major problems of
⇑ Corresponding author.
E-mail address: adomin@cinvestav.mx (A.R. Domínguez-Bocanegra).
of this biomass for generating energy it is vitally important. Pro-
duction of bioethanol from agro-industrial wastes is a suitable
alternative, given the need to replace fossil fuels [4].
Bioethanol is by far the most widely used bio-fuel for transpor-
tation worldwide, because it is a renewable, nontoxic, biodegrad-
able resource and it is oxygenated, there by provides the potential
to reduce particulate emissions in compression–ignition engines
[5]. Second-generation biofuels (Biomass to liquid) are made from
organic materials, such as straw, wood residues, agricultural resi-
dues, reclaimed wood, sawdust, and low value timber. Microorgan-
isms are a key component of the technology used in different
fermentation regimes, including ethanol. Diverse groups of micro-
organisms are capable of producing ethanol [6–8]. These include
yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, bacte-
ria Zymomonas mobilis, fungus Fusariumoxys porum, yeast like fun-
gus Pachysolen tannophylus, and thermophilic bacteria. [9]. S.
cerevisiae and S. pombe represent the organisms of choice for the
industrial production of ethanol due to the following features: they
are capable of fermenting a diverse range of sugars for production
of ethanol under anaerobic. Contamination problem is under

http://dx.doi.org/10.1016/j.fuel.2014.09.062
0016-2361/Ó 2014 Elsevier Ltd. All rights reserved.
86 A.R. Domínguez-Bocanegra et al. / Fuel 149 (2015) 85–89

control as the fermentation process operates at low pH and high
sugar concentration and are genetically stable and ferment 20–
25% (w/v) [5]. Thus, second-generation bioethanol production is
important as it allows improved CO2 balance and make use of
cheap, waste source which does not compete with human food
products. In brief, the use of ethanol as a biofuel is gaining increas-
ing popularity [6]. Although it is produced from several sources but
the technologies using the waste material for its production is most
attractive as it does not interfere with food particular substrates
needed for the ever increasing world population. However, there
are relevant obstacles such as production costs, technology and
environmental problems that need to be overcome in the produc-
tion of second-generation bioethanol [10–14]. The goal of this work
was to investigate the use of raw agro-industrial wastes, namely
coconut milk, pineapple juice and tuna juice, for bioethanol produc-
tion by yeast S. cerevisiae CDBB 790.
Manufacturing Co. Inc., Long Island City, NY) and Absorbance was
taken as in [15].
2.5.3. Sugar determination
The 3,5-dinitrosalicylic acid (DNS) method of Miller [16] was
used to determine residual reducing sugars in the culture media.
A 1-ml sample was centrifuged at 3500g for 5 min, after which
1 ml of DNS reagent was added to the supernatant. The tubes were
covered, heated to boiling point for 5 min and then immediately
placed in an ice-bath for rapid cooling. Then, 8 ml of distilled water
was added. The tubes were shaken using a vortex (model G-560;
Scientific, NY) for 5 min. A spectrophotometer, set at a wave-length
of 575 nm, was employed for optical density measurement and the
data were calibrated with a suitable standard reference curve to
determine the glucose concentration of the samples.

2. Materials and methods 2.5.4. Percentage of ethanol

2.1. Microorganism
S. cerevisiae strain CDBB 790 was obtained from the Microbial
Culture Collection of Mexican CINVESTAV-IPN.
The ethanol content in the samples was measured with a gas
chromatograph Perkin Elmer Autosystem, with a column of HPLC
grade ethanol Zebron FFAP-30 m mark 0–25 min, detector temper-
ature 250 °C, injector temperature 30 °C column temperature 60 °C
for 9 min–10°C/min 200°C–20 min.

2.2. Agro-industrial wastes 2.5.5. Statistical analyses

Raw residual coconut milk was obtained from a local candy
industry located in Mexico City (Col. Vicentina 09340, Delegación
Iztapala) and raw residual tuna juice was obtained from Pachuca,
Mexico and raw residual pineapple juice was obtained from Cen-
tral de Abastos, Mexico. It was sterilized by filtration and no nutri-
ent was added. The chemical composition was utilized gas
chromatograph Perkin Elmer Auto system.
All experiments were performed in triplicate. A tri-factorial
analysis of variance was applied to cell density values of S. cerevisiae
and post hoc comparisons were carried out through the Newman–
Keuls test (P = 0.05); [17]. A similar statistical analysis was made for
bioethanol content; and differences among applied treatments
were determined by means of the post hoc Newman–Keuls test
(P = 0.05).

2.3. Yeast inoculum preparation 3. Results and discussion

The yeast inoculum was grown aseptically in 500 mL Erlen-
meyer flasks containing 250 mL of YM medium (10 g/L glucose,
5 g/L peptone, 3 g/L malt extract, 3 g/L yeast extract), at a constant
temperature of 30 °C and stirrer speed of 150 rpm, for 48 h. Fresh
medium was then inoculated with the seed culture at 10% volume.
The yeast was grown under the same culture conditions.
2.4. Fermentation kinetics
Cultures were grown aseptically in a 500 mL Erlenmeyer flasks
containing 350 mL coconut milk or pineapple juice or tuna juice or
YM medium and inoculated with 35 mL YM medium in the expo-
nential growth phase and incubated using a gyrating shaker
(Gyrating water batch shaker model G76; New Brunswick Scien-
tific, Edison, N.J.) at 150 rpm and 28 °C, for 5 days. Kinetic experi-
ments were carried out in triplicate.
3.1. Chemical composition wastes
The chemical composition of YM medium, coconut milk, pine-
apple juice and tuna juice is shown in Table 1. Total carbohydrates
represent 13–16% of dry mass, including mainly sucrose, which is a
fermentable disaccharide for S. cerevisiae. Indeed, raw juice purity
ranges between 85% and 90% which means that there are about
85–90% of sugars and 10–15% of non-sugars in dry matter. Coconut
milk, pineapple juice and tuna juice, contain significant amounts of
protein 0.33 g/L, 0.5 g/L and 0.5 g/L, respectively and 0.2 g/L lipid
fat for pineapple juice. Such properties, together with a relatively
low price, make of these raw juices very profitable and convenient
materials for ethanol production.
Table 1
Chemical composition YM medium, coconut milk, pineapple juice and tuna juice.

Component YM Coconut Pineapple Tuna

2.5. Analytical methods medium milk juice juice
Carbohydrates total – 16 g/L 13.3 g/L 16 g/L
2.5.1. Dry weight determination Glucose 10 g/L – – –
Culture samples of 2 ml taken from the cultured medium were Yeast extract 3 g/L – –
centrifuged for 5 min at 3500g at room temperature in a model Peptone 5 g/L – – –
5415 centrifuge (Brickman Instruments, NY). The cell pellet was Malt extract 3 g/L – – –
Sodium – 0.25 g/L 0.1 g/L –
washed twice with 2 ml of distilled water and filtered through a
Magnesium – 0.1 g/L – –
0.45-lm Millipore pre-weight filter. The filters containing the bio- Potassium – 2.94 g/L 11.3 g/L 0.34 g/L
mass were dried at 60 °C for 24 h. Chlorite – 1 g/L – –
Protein – 0.33 g/L 0.5 g/L 0.5 g/L
Phosphorus – 11.3 mg/L 7 mg/L 28 mg/L
2.5.2. Growth determination
Lipid fat – – 0.2 g/L –
Yeast cell growth was measured daily by cell microscopic Distilled water 1L
counting using an improved Neubauer haemocytometer (Proper
 A.R. Domínguez-Bocanegra et al. / Fuel 149 (2015) 85–89
Fig. 1. Cells growth of Saccharomyces cerevisiae in different culture media at constant temperature of 30 °C and stirrer speed of 150 rpm rpm (*YM medium;
pineapple juice, tuna juice). Values are means of three determinations.
Fig. 2. Residual reducing sugars of Saccharomyces cerevisiae at constant temperature of 30 °C and stirrer speed of 150 rpm (*YM medium;
tuna juice). Values are means of three determinations.
3.2. Growth S. cerevisiae
In Fig. 1 we can see during the first 12 h the growth S. cerevisiae
is higher in the three agro-industrial wastes that in YM media
(white media); but starting from 15 h the growth of S. cerevisiae
is still increasing in YM medium while in pineapple juice was kept
almost constant. This behavior was different in coconut milk and
tuna juice during the following hours. The maximal growth of
yeast S. cerevisiae occurred at 36 h for almost all the cases; it was
89  106 cel/mL in coconut milk , 57  106 cel/mL in tuna juice,
37  106 cel/mL in pineapple juice and 47  106 cel/mL in YM
medium, (see Fig. 1). In turn, the maximal growth rates (lx) were
0.21 h 1 coconut milk, 0.29 h 1 tuna juice, 0.15 h 1 pineapple juice
and 0.12 h 1 YM medium. According to our results we can clearly
see that the three residues are excellent culture media for growth
yeast S. cerevisiae CDBB790: it was obtained 20–80% more biomass
within tuna juice or coconut milk compared with YM medium,
respectively. It is noteworthy that the growth yeast S. cerevisiae
is lower on pineapple juice probably because some cells are immo-
bilized on the same residue and cannot be counted as free cells.
From Fig. 1, the yeast cells concentration increase was signifi-
cantly different depending on the medium. Covariance analyses
were carried out by means of the Newman–Kuels test. Results
showed that the highest significant growth was recorded by S.
cerevisiae grown in coconut milk, whereas the lowest significant
growth was recorded by S. cerevisiae in tuna juice (P  0.001).
However, the reported growth values of the S. cerevisiae in YM
medium and coconut milk did not show a significant difference
(P > 0.05). In general, yeast cell multiplication occurred during first
87
coconut milk;
coconut milk; pineapple juice,
36 h of the course of fermentation, to reach later some stationary
phase where no significant growth was observed from 40 h to
65 h. Such scenario is certainly related with the rich nutrient com-
position which has been tested to produce secondary metabolites
when used with other yeast species [15].
3.3. Consumption of sugars
In Fig. 2 we can see the consumption of sugars in different
media at 36 h was 88.62% in coconut milk, 93.75% in tuna juice,
90.62% in pineapple juice and 98.6% in YM medium. The maximal
rate of the consumption sugar denoted as lx was 0.052 h 1 coco-
nut milk, 0.07 h 1 tuna juice, 0.057 h 1 pineapple juice and
0.15 h 1 YM medium.
Again with respect to Fig. 2, observe that sugar took 48 h to con-
sume 97.44%, 99.7% and 95.87% of the total sugar, in coconut milk,
tuna juice and pineapple juice, respectively. In the fermentation
the majority of sugar was consumed within 65 h, such that at the
end of the run, 97.62%, 99.5% and 98.75% of the sugar was utilized
within coconut milk, tuna juice and pineapple juice, respectively.
Let us notice that the previous figures are comparatively better
than those reported in similar studies [4,5,18,19].
3.4. Ethanol production
In Fig. 3 we can see that the percentage ethanol produced by the
yeast S. cerevisiae during the first 36 h of fermentation is very
similar in the three residues with 6.5% v/v in coconut milk,
8.39% v/v in pineapple juice and 8.5% v/v in tuna juice; at 36 h and
88 A.R. Domínguez-Bocanegra et al. / Fuel 149 (2015) 85–89
Fig. 3. Ethanol quantification of Saccharomyces cerevisiae at constant temperature of 30 °C and stirrer speed of 150 rpm (*YM medium;
tuna juice). Values are means of three determinations.
48 h important increases were detected to reach the highest ethanol
recorded production of 20% v/v in coconut milk, 22% v/v in pineap-
ple juice and 12% v/v in tuna juice, at 60 h. The maximal rate of eth-
anol production, denoted as lP, was: 0.096 h 1 in coconut milk,
0.1 h 1 in tuna juice, 0.1 h 1 in pineapple juice. According to our
results, no significant differences in the fermentation values were
observed between coconut milk and pineapple juice, moreover fer-
mentation process is still occurring until the end of the course time
at 60 h. Nevertheless, in the case of tuna juice ethanol fermentation
process does not experiment further changes after 48 h.
3.5. Fermentation parameters
In order to make a suitable comparison, two important fermen-
tation parameters (sugar utilization efficiency, ethanol yield) were
considered. The ethanol yield per consumption sugar was 0.435 g/g
in coconut milk, 0.47 g/g in pineapple juice and 0.29 g/g in tuna
juice (see Table 2). The reduction in tuna juice could be due to sev-
eral reasons including the production of compounds other than
ethanol like glycerol, acetic acid and CO2 [20]. There was no signif-
icant difference on the maximal rate of consumption sugar lS as it
was 0.052 h 1 for coconut milk, 0.057 h 1 for pineapple and
0.062 h 1 for tuna juice which is related with the fact that at
48 h of time course sugar consumption was higher than 96% in
all the cases. Results presented here confirmed that biocatalyst
process of yeast cells on coconut milk or pineapple juice for etha-
nol fermentation was at least as efficient as in other widely used
materials like molasses or thick juice [4,5,18]. Moreover, ethanol
fermentation on coconut milk or pineapple juice has been found
to be economically favorable in terms of all presented process
parameters and also considering that no additional sugar was
required. It is necessary to attain high ethanol concentration in
order to decrease the costs of ethanol distillation [21].
It is clear that ethanol synthesis was partially associated with
biomass growth and still occurred even after growth stopped and
Table 2
Significant fermentation parameters obtained during the fermentation of coconut
milk, pineapple juice and tuna juice by yeast S. cerevisiae.
Media Initial sugar, Consumption Ethanol yield
So (g/L) sugar Ys (%): per consumption
Ys/x (%) sugar Yp/s (g/g)
Coconut milk 17.6 98.75 0.435
Tuna juice 13 99.5 0.29
Pineapple juice 16 97.62 0.47
coconut milk; pineapple juice,
sugars were exhausted (Figs. 2 and 3). Indeed, both the maximal
growth and ethanol production occurred in coconut milk
89  106 cel/mL, with 22% v/v of ethanol, see Figs. 1 and 3. This fig-
ure was followed by the one present in pineapple juice with
37  106 cel/mL maximal growth and 20% v/v of ethanol. Notice
that, tuna juice offered a nice maximal growth of 57  106 cel/mL
but it exhibited just 12% v/v of ethanol production. Nevertheless,
in the case of tuna juice ethanol fermentation process does not
experiment further changes after 48 h. Clearly the previous results
are in correspondence with the above mentioned ethanol yield per
consumption sugar.
The increase of ethanol concentration in coconut milk and pine-
apple with respect to tuna juice was probably due to the presence
of rich nutrient composition (potassium and phosphorus) in the
above mentioned raw juices, [22]. Notice that, the observed fer-
mentation values are in the magnitude order as the ones given
with molasses or thick juice, [4,5,21] and also correspond well to
report values for immobilized cells were free cells fermentation
values were given as initial reference figures in [19].
4. Conclusions
The agro-industrial wastes coconut water, pineapple juice and
tuna juice are excellent culture media for growing the yeast S. cere-
visiae CDBB 790 without other nutrient addition.
In this paper it was verified that raw coconut water, raw pine-
apple juice and raw tuna juice are excellent media for the produc-
tion of bioethanol by the yeast S. cerevisiae. Their physicochemical
properties, availability and low cost of these agro-industrial wastes
makes them highly competitive substrates of second generation for
ethanol production and reduction environmental pollution.
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