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Producción de bioetanol a partir de cáscaras de yuca (Manihot esculenta)
Bioethanol production from cassava (Manihot esculenta) peels
Resumen
En los últimos años, la producción de etanol a partir de la abundancia de biomasa de celulosa de bajo costo o de residuos
agrícolas ha crecido en importancia, debido a la esperanza de reducir el costo de la producción de etanol y beneciar el medio
ambiente global. La aplicación del uso de residuos de yuca para la producción de etanol podría ser de gran ventaja para la
economía de un país; por lo tanto, este estudio se llevó a cabo para determinar la posibilidad de la producción de bioetanol a
partir de cáscaras de yuca como una fuente más barata de bioetanol. Las cáscaras de yuca fueron recolectadas, limpiadas,
picadas y fermentadas durante 14 días por Saccharomyces cerevisiae aislado del vino de Palma. En este estudio se analizaron
parámetros que incluyeron biomasa, rendimiento de etanol, pH, acidez titulable y azúcar reductora. Se observó que hubo una
disminución en el pH de 5,0 a 3,8 en el lote de fermentación mejorado con levaduras con rendimiento de etanol de 7,5 ml, y
aproximadamente 8,1% de contenido de alcohol. Presentó un aumento progresivo de la acidez valorable y de la biomasa celular;
y una disminución en la reducción del azúcar durante el curso de la fermentación de los lotes de ensayo y control. Los resultados
de esta investigación, demostraron que la producción de etanol a partir de cáscaras de yuca, podría dar solución a los problemas
de su eliminación en el medio ambiente y también servir como una opción alternativa a la producción de etanol, a partir de
materias primas disponibles más baratas.
Palabras Clave: cáscaras de yuca; bioetanol; fermentación; Saccharomyces cerevisiae.
Abstract
In recent years, the production of ethanol from plentiful, low cost cellulosic biomass or agricultural wastes has grown in importance
due to the hope that it could reduce the cost of ethanol production and benet the global environment. The application of using
cassava residues for ethanol production could be of great advantage to a country’s economy; hence, this study was carried out
to determine the possibility of bioethanol production from cassava peels as a cheaper bioethanol source. Cassava peels were
collected, cleaned, chopped and fermented for 14 days by Saccharomyces cerevisiae isolated from palm wine. In this study,
parameters including biomass, ethanol yield, pH, titratable acidity and reducing sugar were analyzed at two day intervals using
standard methods. There was a drop in pH from 5.0 to 3.8 in the yeast ameliorated batch of fermentation with ethanol yield of
7.5 mL and about 8.1% alcohol content produced. There was a progressive increase in titratable acidity and cell biomass; and
a decrease in reducing sugar during the course of fermentation of both the test and control batches. The results from this study
showed that ethanol production from cassava peels could provide solution to the problems of their disposal into the environment
and also serve as an alternative option to ethanol production from cheaper available raw materials.
Keywords: cassava peels; bioethanol; fermentation; Saccharomyces cerevisiae.
*Chinwe-Christy, Isitua
1
; Scholastica-Onyebuchi, Anadozie
2
; Isaiah-Nnanna, Ibeh
3
(Recibido: Febrero 2018, Aceptado: Mayo 2018)
1
Department of Biological Sciences, Microbiology Program, College of Sciences, Afe Babalola University Ado-Ekiti, P.M.B.
5454 Ekiti State, Nigeria. Email: christykings@yahoo.com, isituacc@abuad.edu.ng
2
Department Chemical Sciences, Biochemistry Program, College of Sciences, Afe Babalola University Ado-Ekiti, P.M.B. 5454
Ekiti State, Nigeria.
3
Department of Medical Laboratory Sciences, School of Basic Medical Sciences, University of Benin, P.M.B. 1154, Benin City,
Edo State, Nigeria.
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INTRODUCTION
Bioethanol is a principal fuel that can be used as
petrol substitute for vehicle. It is a renewable energy
source produced mainly by sugar fermentation
process, although it can also be manufactured
by the chemical process of reacting ethylene with
steam. The main sources of sugar required to
produce ethanol come from fuel or energy crops
like cassava and cassava products, waste straw,
sawdust, etc. (1).
Yeast (Saccharomyces cerevisiae), have been
known to humans for thousands of years as they
have been used in fermentation processes like in
the production of alcoholic beverages (2) and bread
leavening (3). Yeasts metabolize sugar to produce
ethanol and carbon dioxide. The basic carbon and
energy source for yeast culture are sugars (2) (3).
Cassava (Manihot esculenta), also known as
manioc, tapioca or yucca, is one of the most
important food crops in the humid tropics, being
particularly suited to conditions of low nutrients
availability and is able to survive drought (4). It is
the third largest source of carbohydrates for human
consumption in the world, with an estimated annual
world production of 208 million tonnes (5). The
major harvested organ is the tuber, which is actually
swollen root. The nutrient reserve of cassava
is made up of starch. Cassava peels is gotten
during the processing of the cassava tuber and
it is an agricultural waste. Cassava peels contain
starch which when treated with a varying level of
H2SO4 undergoes an abrupt change in the physical
structure of the glycosidic bond linking amylase
and amylopectin. Glycosidic bond are broken to
produce glucose and oligosaccharide residues (4).
In Africa, especially Nigeria, which is one of the
largest centre of cassava production, it is grown on
7.5 million hectares of land and produces about 60
million tonnes per year. Thus, wastes (especially
cassava peels) generated from the processing
of cassava into various products are littered or
dumped in the environment causing pollution. There
is therefore the need for revalorization of cassava
peels waste into useful products. The application of
using cassava peels for ethanol production could
be of great advantage to a country’s economy.
This study therefore determines the possibility of
bioethanol production from cassava peels which
could provide a cheaper bioethanol source; also,
exploit the fermentative ability of Saccharomyces
cerevisiae isolated from palm wine in the production
of the desired bioethanol.
MATERIALS AND METHODS
Collection and processing of cassava peels
Cassava peels were obtained from a cassava
milling factory in Uselu market, Benin City, Edo
State, Nigeria. The peels were washed in clean
water (to remove sand and cyanide content) and
weighed on a laboratory scale. Thereafter, the
peels was allowed to dry naturally (de-watering)
for 4 hours on a clean tray, after which they were
chopped into bits and transferred into a mortar
where they were mashed using a pestle to attain
sufcient size reduction. This was to ensure the
creation of sufcient surface area of the material to
aid the process of fermentation.
Isolation and identication of yeast
(Saccharomyces cerevisiae) from palm wine
Yeast used for this experiment was isolated from
fermented palm wine. One mL of the serially
diluted palm wine sample was plated on sabouraud
dextrose agar supplemented with streptomycin
(0.05 mg/L) using pour plate method and incubated
at 28oC for 48 hours. The yeast colonies that
developed were isolated and puried by spread
plate method on fresh sabouraud dextrose agar
plates. Identication of the yeasts was by the use
of standard morphological and physiological tests
and identication keys described by Barnett et al.
(6) (14).
Preparation of sample for fermentation
100g of the mashed cassava peel was transferred
into two different 1L fermentation asks and 1000mL
of distilled water was added to each of them. The
asks were autoclaved at 121oC for 15 minutes
and allowed to cool. The contents of the asks
were then ltered using a muslin cloth to obtain the
desired cassava medium and again autoclaved and
allowed to cool. For hydrolysis to form sugars, 5mL
of 5% H2SO4 and 5mL of 5% NaOH were added
to each jar and heated to about 50oC. Thereafter,
20mL suspension of the inoculum (yeast) was
introduced aseptically into one of the fermentation
asks which served as the test experiment, while
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the other fermentation ask had no inoculum and
served as the control. The asks were corked with
a rubber stopper and left to ferment for 14 days.
Certain parameters of the samples were analysed
at 2 day intervals for the period of fermentation.
Biochemical parameters determined during
fermentation
Samples obtained from the fermentation asks were
centrifuged and the supernatants were examined
for various parameters including; pH, temperature,
total reducing sugars, ethanol yield, cell biomass
and titratable acidity.
Measurement of pH value
The pH values of each fermented medium was
determined with the aid of a pH meter after
standardization with buffer at pH 5. This was done
by dipping the electrode into the different samples
in the ask and readings taken before and after
fermentation.
Measurement of temperature
Temperature values of each fermented medium
was determined by the use of a thermometer before
and after fermentation.
Titratable acidity determination
Fermented media were analysed for titratable
acidity using phenolphthalein as indicator. This was
carried out using the methods of Isitua and Ibeh
(2); Association of Analytical Communities, AOAC
(7) by titrating 10mL of sample against 0.1M NaOH
using the rst permanent colour change.
Assay for reducing sugar content
The total reducing sugar content in the samples
were determined using 3, 5-dinitrosalicylic acid
(DNS) reagent. Briey, 3 mL of DNS reagent was
added to 3 mL of each of the samples and then, test
tubes were tightly caped to avoid loss of liquid due
to evaporation. Test tubes contents were heated
at 90oC for 5-15 minutes to develop a red/brown
colour. 1 mL of a 40% potassium sodium tartrate
(Rochelle salt) was added to stabilize the colour.
After cooling to room temperature in cold water
bath, absorbance was read in a spectrophotometer
at 600 nm wavelength against reagent blank (7).
Cell biomass determination
Cell biomass of the fermented media was
determined by measuring the optical density of
each fermentation media at 600 nm. The aliquots
(about 2.5 mL) of the different fermentation
media were transferred into a 3 mL cuvette and
absorbance were read against a reagent blank. The
reagent blank was prepared with the uninoculated
cassava medium (control) in place of the inoculated
cassava medium. This analysis was to determine
the turbidity in each fermentation medium during
the fermentation process (7).
Determination of the amount of alcohol
produced
The amount of alcohol (% v/v) produced in each
of the fermentation medium after the period of
fermentation was determined according to the
method described by Isitua and Ibeh (2) from
values of their respective specic gravities obtained
by using a hydrometer and calculated as follows:
Alcohol content (% v/v) = (D1 – D2) x 105 x 1.25
Where D1 = 1.06
D2 = Mean of hydrometer readings for each
fermentation sample.
To obtain the ethanol yield, the fermented cassava
medium was ltered using cheese cloth to obtain
liquid for distillation. Distillation involved separating
ethanol from water and other residual solids after
fermentation. Ethanol was boiled off from the rest of
the solution in a distillation column. The condensed
ethanol and water were allowed to travel through a
rectier column where the water remaining in the
ethanol was nally removed. This was carried out at
a temperature of about 78.5oC. Ethanol yields after
distillation were measured after 10 minutes for the
cassava medium containing S. cerevisiae as well
as the control experiment (8).
Statistical analysis
The assays were carried out in triplicate; the
results were mean values ± standard deviation and
expressed as mg / mL of sample. The amount of
alcohol produced was expressed in % v/v of the
fermented medium.
RESULTS
During the fermentation, the amount of ethanol
yield, ethanol concentration, cell biomass ( Figure
1), pH, temperature, reducing sugar and titratable
acidity were determined and the results are shown
in Figure 2 (a, b, c, d). The ethanol yield and
concentration after distillation of fermented cassava
peels medium ranged from 2.2 mL to 7.5 mL and
Isitua et al. Bioethanol production from cassava (Manihot esculenta) peels pp. 40-45
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CMS= Cassava medium with S. cerevisiae. CM= Cassava medium without
S. cerevisiae
Figure 1. Ethanol yield and concentration during
fermentation period of cassava peels
CMS= Cassava medium with S. cerevisiae. CM= Cassava medium without S. cerevisiae
Figure 2. (a) pH values; (b) temperature (oC); (c) titratable acidity; (d) reducing sugar during fermentation
period of cassava peels.
2.3% v/v to 8.1% v/v respectively for the cassava
medium containing S. cerevisiae (test sample);
while it was from 1.8 mL to 2.0 mL and 1.8% v/v to
2.0% v/v respectively for the control (Figure 1).
There was a decrease in pH (from 5.0 to 3.8)
during the fermentation period, though within
acidic range (Figure 2a), while the temperature
varied throughout the fermentation period; ranging
from 28.0oC to 33.0oC for test sample (Figure
2b). Titratable acidity increased all through the
fermentation period (Figure 2c) with a decrease in
reducing sugar (Figure 2d), as well as an increase
in cell biomass (Figure 3).
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DISCUSSION
Over the years, the peels obtained from the
processing of cassava are disposed off as
agricultural wastes constituting nuisance and
eyesore in the environment. Consequently, a
large amount of cassava peels waste is generated
annually (9), (10), (11). In developed countries,
substrates such as corn, sugarcane and beets
have been used in the production of ethanol. In this
study, we have explored the utilization of readily
available wastes as cassava peels in the production
of ethanol.
Ethanol yield after distillation of fermented cassava
medium containing yeast isolates during the
fermentation period was higher than the ethanol
yield from the control experiment (Figura 1). This
high ethanol yield from the test sample could be
attributed to the presence and fermentative activity
of the yeast S. cerevisiae; and this ndings is similar
to result obtained by Srinorakutara et al. (8) who
reported a range of 3.5ml to 12.0ml for ethanol yield
from cassava wastes. The successful production of
ethanol from cassava peels (wastes) have shown
that these waste contain fermentable material
which was evident from the increase in ethanol
yield throughout the fermentation period (2). The
ethanol produced was recorded for all experimental
days except day 0, and the values recorded show
that ethanol production from cassava medium with
yeast had the highest value of 7.5ml as at day 14
and the least value of ethanol yield was recorded
on day 2 as 2.2ml.
There was a decrease in pH during the fermentation
period, though within acidic range, suggesting
that the fermenting microorganism (S. cerevisiae)
must have the inherent capacity to tolerate acidic
condition. The pH values ranged from 5.0 to 3.8 in
the test medium and 5.0 to 4.8 in the control medium.
This decrease in pH values could be attributed to
the formation of organic acids during fermentation
(12). The variability in temperature throughout the
fermentation period indicate that heat is released
during fermentation; while the increase in titratable
acidity may be due to increase in acid production
in the fermentation media. The reducing sugar of
the test sample declined gradually throughout the
fermentation period, and this may be due to the
bioconversion of the fermentable sugar present
to ethanol by S. cerevisiae (2), (13), (14). For the
test sample, the cell biomass increased till day 10
and started declining slightly until the end of the
fermentation period.
CONCLUSION
The various important uses of ethanol and the
importance of ridding the environment of the
harmful effects due to piling up of agricultural waste
products, such as cassava peels, underscore the
signicance of this study. The results obtained from
this experiment revealed that fermentable material
is present in a reasonable amount in cassava
peels waste. If these peels waste are fermented
under stipulated experimental conditions using
S. cerevisiae, a substantial amount of ethanol
which can be used as chemical feedstock will be
produced. Thus, the importation of ethanol can be
reduced if substantial resources are devoted to the
production of ethanol from cassava wastes.
CONFLICT OF INTEREST
Authors have declared no conict of interest.
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medium without S. cerevisiae
Isitua et al. Bioethanol production from cassava (Manihot esculenta) peels pp. 40-45
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