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PRODUCTION OF BIO-ETHANOL FROM POTATO STARCH WASTES BY

Saccharomyces cerevisiae

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Egypt. J. of Appl. Sci., 34 (12) 2019 256-267

PRODUCTION OF BIO-ETHANOL FROM POTATO

STARCH WASTES BY Saccharomyces cerevisiae
Taha, M.G. ; A.E. Khattab ; H.E. Ali and M.A.M. Dawood
Agric. Biochem. Dept., Fac. Agric., Al-Azhar Univ., Cairo Egypt.
Key Words: acid hydrolysis, potato starch wastes, bio-ethanol
production, Saccharomyces cerevisiae.
ABSTRACT
Bio-ethanol is one of the energy sources that can be produced by
renewable sources. Potato starch wastes were chosen as a renewable
carbon source for ethanol fermentation because it is relatively
inexpensive compared with other feedstock considered as food sources.
However, saccharification processes are needed to convert starch of
potato into fermentable or reducing sugars before ethanol fermentation.
In this study, hydrolysis of potato starch wastes and growth parameters of
the ethanol fermentation were optimized to obtain maximum ethanol
production by S. cerevisiae S288c. The ratio of plant material to acid
solution of 1:10 (w/v). Results demonstrated that 0.5% H2SO4, 1%
H2SO4, 2% H2SO4 and 3% H2SO4 at 121ºC for 20 min by autoclave were
enough to hydrolyze all starch contained in the potato starch wastes. The
maximum yield of reducing or fermentable sugars was 125.8 mg/g
obtained in 0.5% H2SO4. The minimum yield was 53 mg/g obtained in
3% H2SO4. The yield of bioethanol production by S. cerevisiae S288c
was (51.37 mg/g) was achieved at pH 5.5, temperature of 30⁰C and
inoculums size of 10% (v/v) after 72 hours of fermentation.
INTRODUCTION
Increasing industrialization and the population has led to a
continuous rise in global energy demand. At present, more than 80% of
world energy production of fossil fuel use. However, the depletion of
fossil fuels at an alarming rate, the causes of environmental pollution and
burned (Láinez, et al., 2019). Therefore, there is a need for sustainable
and renewable energy sources that do not affect the environment and
ecosystems. Biofuels have emerged recently fuel tankers are ideal to
meet the energy needs in a sustainable manner (Morais, et al., 2019).
More specifically, can be used as an alternative oil sources bioethanol
and has become one of the most dominated biofuels industry because the
majority of the emissions of carbon dioxide, which contributed to the
transport sector. In addition, ethanol has been renovated high energy
oxygen content easily stored Zhang, et al., 2019).
Agricultural and industrial, such as starchy substrates, waste and
high availability has shown, and biological degradation which rich in
nutrients. in addition, its use in the production of bio-fuel operations
removes waste disposal problems. A variety of agro-industries raw

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257 Egypt. J. of Appl. Sci., 34 (12) 2019
materials can be used as substrates for biological conversion of ethanol.
Waste crop tubers such as potatoes, sweet potatoes and cassava substrates
are favorable because they contain enough amounts of starch, which can
be hydrolysed to sugars and later fermented to ethanol (Lin, et al., 2010).
Potatoes are especially suitable because of its high return carbohydrate
fermented (Lantero, et al., 2011). Furthermore, potatoes are the third
most important food crop in the world after rice and wheat, which are the
basic crops. Ratio widespread use in the fields of industry and large
quantities of waste potato peel (PPW) are created. Manufacturing
potatoes produced between 20 and 50% of the waste of raw product
(Rezig, et al., 2010).
Starchy materials require a reaction of starch with water
(hydrolysis) to break down the starch into fermentable sugars
(saccharification). Hydrolysis is carried out at high temperature (90 to
110°C); however, at low temperatures, it is also possible and can
contribute to energy savings (Sanchez, et al., 2008). To convert starch
into the fermentable sugars, either acid hydrolysis or enzymatic
hydrolysis needs to be performed. Each has their own set of advantages
and disadvantages for use. Enzyme hydrolysis is generally chosen even
though high cost of enzymes and initial investment because of high
conversion yield of glucose (Tasić, et al., 2009).
Therefore, this investigation was carried out to study utilization of
potato starch waste as a very cheap substrate for the production of Bio-
ethanol by Saccharomyces cerevisiae S288c.
2. MATERIALS AND METHODS
2.1- Materials:
The potato starch wastes (PSW) were collected from a chips
factory for the food industries (Egypt Foods Company, Quesna,
Menoufia, Egypt). It was dried at 50◦C for 48 hours, ground and sieved to
get particles with particle size between 500 and 1000 µm. it was stored at
room temperature (25 ± 5◦C) until use.
2.2- Chemicals and Reagents:
Chemicals and reagents of the analytical methods used in present
study were sulfuric acid, sodium hydroxide, glucose, Benedict’s reagent,
dinitrosalicylic acid reagent (DNS reagent), sodium sulphite, Rochelle
salt (potassium sodium tartrate), phenol, yeast extract, malt extract,
sodium chloride, peptone, agar, dipotassium hydrogen phosphate,
potassium dihydrogen phosphate, magnesium sulfate, ammonium sulfate,
sodium hydroxide and potassium dichromate. They purified and distilled
before use. All chemicals were purchased from El- Gamhouria Trading
Chemicals and Drugs Company, Egypt.

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Egypt. J. of Appl. Sci., 34 (12) 2019 258
2.3- Micro-organisms
S. cerevisiae S288c was obtained from Microbial Biotechnology
Department, National Research Center (Dokki, Egypt), it was used in this
study and maintained on yeast extract-malt extract (YM) agar slant at
4oC.
2.4 - Analytical methods:
2.4.1- Chemical composition of potato starch wastes:
The chemical composition of potato starch wastes (PSW) were
determined according to Zhao et al., (2005) by Near-Infra Red (NIR)
Spectroscopy apparatus, model DA1650, which manufactured by FOSS
Corporation. (NIR) spectrometer at wavelength region from (750-2500
nm) of the electromagnetic spectrum, which used to analyze the chemical
structure and to take a fingerprint.
2.4.2- Acid Hydrolysis or Saccharification:
The PSW (25g ) was dried at 80ᵒC. H2SO4 0.5%, 1 %, 2% and
3% (w/v) 1:10 solid-liquid ratio was added to the samples powder, the
mixtures were then autoclaved at 121ᵒC for 15 min. Finally, the samples
were cooled down and analysed for glucose concentration according to
Sheikh et al., (2016).
2.4.3- Determination of reducing sugars:
2.4.3.1- Qualitative analysis of reducing sugars:
A biochemical test to detect reducing sugars in solution, devised
by the US chemist S. R. Benedict (1884–1936). Benedict's reagent a
mixture of copper (II) sulphate and a filtered mixture of hydrated sodium
citrate and hydrated sodium carbonate is added to the test solution and
boiled. A high concentration of reducing sugars induces the formation of
a red precipitate; a lower concentration produces a yellow precipitate.
Benedict's test is a more sensitive alternative to Fehling's test.
2.4.3.2- Quantitative analysis of reducing sugars:
Total reducing sugars in the hydrolysate of potato starch wastes
were estimated by the dinitrosalicylic acid (DNS) colorimetric method
adapted from previous work (Miller, et al 1961 and Ghose, 1987).
2.4.3.2.1- Preparation of standard solution
The standard glucose stock solution 10 g/L was prepared by
dissolving 0.20 g of D-(+)-Glucose anhydrous (C6H12O6) in 20 mL of
distilled water. Working solutions were daily prepared by appropriate
dilution of the stock solution in DI water.
2.4.3.2.2 Preparation of dinitrosalicylic acid reagent
3,5-dinitrosalicylic acid reagent was prepared by dissolving 1 g of
3,5-dinitrosalicylic acid in 20 mL of 2 M NaOH. It was then mixed with
potassium sodium tartrate (C4H4KNaO6) solution (30 g of C4H4KNaO6 in
50 mL of distilled water) on a magnetic stirrer hot plate and diluted to
100 mL with distilled water.

3
259 Egypt. J. of Appl. Sci., 34 (12) 2019
2.4.3.2.3- Calibration curve:
Calibration curve for estimation of reducing sugar yield was
obtained by plotting the absorbance (at 520 nm) vs. concentrations of
standard glucose in the range of 0.20-1.00 g/L. The concentrations of
glucose were daily prepared by dilution of the stock solution.
2.4.3.2.4- Estimation of reducing sugar yield in the hydrolysate of
potato starch wastes.
0.50 mL dinitrosalicylic acid was introduced into a test tube
containing 0.50 mL of standard glucose or the hydrolysate of cellulose. It
was then boiled at 100oC for 10 min and cooled in an ice bath.
Afterward, 5 mL distilled water was added, shaken and left for 5 min.
The absorbance was measured at 520 nm against reagent blank using a
UV-Visible spectrophotometer (Nicolet- evolution300- Thermo Electron
Corporation). To calculate the quantitative of reducing sugar yield in the
form of g/100 g substrate, the following equation was used:
Reducing sugar yield (g/100 g substrate)
Where RC is the reducing sugar concentration (g/L), V1 is the volume of
acid solution (mL), and M1 is the mass of substrate added (g).
2.5-Fermentation process:
2.5.1- Preparation of inoculums medium:
S. cerevisiae S288c was activated on yeast extract-malt extract
(YM) agar plates containing (per L): 3 g yeast extract, 3 g malt extract, 5
g peptone and 10g glucose. It was then incubated at 30oC for 24 h,
streaked into YM broth (pH ≈ 5.5) (all ingredients like the Petri dishes of
YM agar, except agar powder) and incubated again at 30oC for 24 h,
according to Pridham et al., (1957).
2.5.2- Fermentation medium and conditions:
The acid hydrolysate of the starch under the appropriate
conditions was adjusted to pH ≈ 5.5 using 2 M NaOH, supplemented
with following additional nutrients (per L): 1 g yeast extract, 1 g
MgSO4.7H2O, 2 g (NH4)2SO4 and 5 g KH2PO4 (Akaracharanya, et al.,
2011) and then used as an ethanol production medium. This medium with
a working volume of 100 mL was transferred into a 250 mL Erlernmeyer
flask and sterilized by autoclaving at 121oC, 15 psi for 30 min. Then, an
inoculums suspension of S. cerevisiae S288c cells was loaded into the
sterilized medium (10% v/v). The fermentation was operated at 30oC
under static conditions for 72 h. The fermented broth was collected at 6-h
time intervals for analysis of ethanol concentration.
2.6.-Estimation of bioethanol:
2.6.1-Qualitative estimation
Bioethanol production was examined by Jones reagent
(K2Cr2O7+H2SO4; Jones 1953). One milliliter of K2Cr2O7 (2 %), 5 ml of

4
Egypt. J. of Appl. Sci., 34 (12) 2019 260
H2SO4 (concentrated) and 3 ml of sample were added to Jones reagent.
Ethanol was oxidized into acetic acid with potassium dichromate in the
presence of sulfuric acid and gave blue-green color. Green color indicates
positive test (Caputi, et al., 1968).
2.6.2- Quantitative estimation:
Ethanol production was estimated according to Doelle and
Greenfield, (1985) by detecting the ethanol concentration in each sample
after fermentation under all biochemical conditions with Gas
chromatography (model 6890, Agilent), equipped with flame ionization
detector and nominal capillary column (60 m×530 µm ×5.00 µm).
Helium was the carrier gas, flow rate was 25 mL/min. Oven and detector
temperature was 300ºC. The theoretical ethanol yield was calculated
assuming the conversion of all the hydrolysed sugars at the end of the run
to ethanol.
3- RESULTS AND DISCUSSION
3.1- Chemical composition of potato starch wastes:
Data in Table (1) shows the chemical composition of potato
starch wastes. The moisture content recorded that 14.13%, the fat content
1.60%, the crud protein 1.33%, fiber content 0.18%, ash content 1.21%
and the total carbohydrate 82.88%. Results indicate that the potatoes
wastes are very rich of carbohydrates, which represent an important
source in the production of bioethanol.
These results agreement with Khawla, et al., (2014) mentioned
that the characterization of potato peel waste (PPW) contained (on dry
basis) proteins (15.1 ± 0.8%, w/w), fat (0.52 ± 0.09%,w/w), moisture
(6.78 ± 0.22%, w/w), starch (48.46 ± 1.88%) and ashes (7.2 ± 0.2%,
w/w). Arapoglou, et al. (2010) said that the chemical composition of
potato starch wastes were (6.34% ash content, total carbohydrate 68.7%,
8% proteins and fat 2.6%). Sheikh, et al., (2016) reported that the
amount of moisture and ash content of potato peels wastes (PPW) are
7.50 % and 7.71 % respectively. Liang, et al., (2014) observed chemical
composition of potato peel waste (PPW) were carbohydrate 63.2% ± 4.2,
starch 34.3% ± 2.7, protein (N tot 6.25) 17.1 % ± 0.3 and lipids 1.2 % ±
0.0. Rani, et al., (2010) mentioned that the potato flour were 8.12%
moisture, 73.0% starch, 10.86% total protein, 1.65% crude fiber, 2.15%
ash content, 1.00% total lipids. Duhan, et al., (2013) reported that the
potato flour contained 8.39% moisture, 73.25% starch and
4.86%proteins.

5
261 Egypt. J. of Appl. Sci., 34 (12) 2019
Tab. (1): Chemical composition of potato starch wastes (Average).
Parameters Dry weight (%)
Moisture Content (%) 14.13
Fat Content (%) 1.60
Crude Protein (%) 1.33
Fiber Content (%) 0.18
Ash Content (%) 1.21
Total Carbohydrate (%) 82.88
3.2- Determination of reducing sugars:
3.2.1- Qualitative analysis of reducing sugars:
Table (2) shows the qualitative analysis of reducing sugars after
acid hydrolysis or (scarification) of potato starch wastes by using
Benedict's test. The sample A (H2SO4 0.5%) a red precipitate appeared,
sample B (H2SO4 1%) with a red precipitate appeared, sample C (H2SO4
2%) it showed up an orange precipitate, sample D (H2SO4 2%) an orange
precipitate appeared. These results according to Benedict's test.
Tab. (2): Qualitative analysis of reducing sugars by Benedict's test
Benedict's Reagent
Samples H2SO4 (%)
Before after
Sample A 0.5% Blue color Red precipitate
Sample B 1% Blue color Red precipitate
Sample C 2% Blue color Orange precipitate
Sample D 3% Blue color Orange precipitate
3.2.2- Quantitative analysis of reducing sugars:
Miller (1959) reported the dinitrosalicylic acid (DNS) was used
to test for the present of free carbonyl group (C=O) present in reducing
sugars. The aldehyde group in reducing sugars was reduced with 3,5-
dinitrosalicylic acid to form 3-amino,5-nitrosalicylic acid, which absorbs
light strongly at 520 nm, as presented in Figure (1).

Fig. (1). Chemical reaction of glucose with 3,5-dinitrosalicylic acid (Miller, 1959)

6
Egypt. J. of Appl. Sci., 34 (12) 2019 262
It was first introduced as a method to detect reducing substances
in urine and has since been widely used, for example, for quantification
of carbohydrate levels in blood. It is mainly used in assay of alpha
amylase. However, enzymatic methods are usually preferred to DNS due
to their specificity.
The determination of reducing sugars by a UV-Visible
spectrophotometer by used DNS method for estimation of total reducing
sugar yield is presented in table (3).
Tab. (3): estimation of total reducing sugar yield (mg/gm) by a UV-
Visible spectrophotometer.
Samples H2SO4 (%) Total reducing sugar (mg/gm)
Sample A 0.5 125.8
Sample B 1 91.4
Sample C 2 67.6
Sample D 3 53.5
This table observed the dilute H2SO4 break down starch and turns
it into reducing sugar in the form of glucose. The results revealed that
reducing sugar yield increases from 53.5 to 125.8 mg/gm when H2SO4
concentrations decrease. The maximum yield of total reducing sugar
yield 125.8 mg/g was obtained in 0.5% H2SO4. The minimum yield of
total reducing sugar yield 53.5 mg/g was obtained in 3% H2SO4, because
sulfuric acid in the case of increased concentration removes a molecule
of water from glucose and converts it to Hydroxy methyl furfural (HMF)
compound. Hydroxymethylfurfural (HMF) is a product of dehydration of
hexose sugars such as glucose, fructose and carbohydrates, as presented
in Figure (3).

Fig. (2): Formation of HMF from D-glucose (Tao et al., 2011)

These results agreements with Hashem and Darwish (2010)

mentioned that the potato starch residue stream produced during chips
manufacturing was used as an economical source for biomass and
bioethanol production by Saccharomyces cerevisiae. Results

7
263 Egypt. J. of Appl. Sci., 34 (12) 2019
demonstrated that 1% H2SO4 at 100 ºC for 1 h was enough to hydrolyze
all starch contained in the residue stream.
3.3- Estimation of bioethanol:
3.3.1- Qualitative estimation
After fermentation process bioethanol production was examined
by Jones reagent (K2Cr2O7+H2SO4; Jones 1953). One milliliter of
K2Cr2O7 (2 %), 5 ml of H2SO4 (concentrated) and 3 ml of sample were
added to Jones reagent. Ethanol was oxidized into acetic acid with
potassium dichromate in the presence of sulfuric acid and gave blue-
green color. Table (4) shows the results of bioethanol reaction with Jones
reagent. It showed yellow color with control and sample before
fermentation but it was green color with sample after fermentation.
Tiwari, et al., (2015) mentioned that the bioethanol production was
examined by Jones reagent. Ethanol was oxidized into acetic acid with
potassium dichromate in the presence of sulfuric acid and gave blue-
green color. (Caputi, et al., 1968) reported that the ethanol with Jones
reagent was green color indicates positive test.
Tab. (4): Qualitative estimation of bioethanol by Jones reagent.
Group Treatments Jones reagent
1 Control -Yellow color
2 Sample before fermentation -Yellow color
3 Sample after fermentation + Green color
3.3.2- Quantitative estimation:
Ethanol yield by GC and productivity obtained by
fermentation of the hydrolysate are presented in Table (5). In this
study, the hydrolysis of starch was performed using dilute H2SO4
further fermented to ethanol using S. cerevisiae S288c and it was
found that the ethanol yield of 51.37 mg/g substrate corresponded
to a productivity ethanol yield of 0.75(mg/g/ h).
These results agreements with Mahmoodi, et al., (2018)
indicated that the ethanol yield of 44.6 and 44.4 g per 100 g glucose was
obtained from hydrolysate and acid treatment liquor, respectively.
Mushimiyimana and Tallapragada (2016) observed that the alcohol
content was 15.34 % and 14.4 % by Gas chromatography by using potato
peel and onion peel as substrates. Tasić, et al., (2009) showed that the
ethanol yield of starch from fresh potato tubers of 31 g/L was obtained in
the fermentation of hydrolyzate prepared under the optimal hydrolysis
conditions by commercial bakery yeast at 28 ◦C for about 18 h.
Izmirlioglu and Demirci (2012) they said that the maximum bio-ethanol

8
Egypt. J. of Appl. Sci., 34 (12) 2019 264
production from waste potato mash by using Saccharomyces Cerevisiae
was obtained at the optimum conditions of 30.99 g/L ethanol.
Tab. (5): Ethanol yield and productivity obtained by fermentation of
the starch hydrolysate.
Initial glucose Optimum Maximum ethanol Ethanol
concentration (mg/g fermentation time yield (mg/g productivity
substrate) (h) substrate) (mg/g/ h)
125.8 72 51.37 0.71
Under anaerobic conditions, the pyruvate is further reduced to
ethanol with the release of CO2. Theoretically, the yield is 0.511 for
ethanol and 0.489 for CO2 on a mass basis of glucose metabolized, as
represented by the following Eq. (Zhang, et al., 2010)

C6H12O6 2CH3CH2OH + 2 CO2

Glucose 1g Ethanol 0.51g Carbon dioxide 0.49g
CONCLUSION
The hydrolysis of potato starch wastes by mineral acids was
studied. Using 0.5% H2SO4, 1% H2SO4, 2% H2SO4 and 3% H2SO4 at
121ºC for 20 min by autoclave the ratio of plant material to acid solution
of 1:10 (w/v), the highest fermentable or reducing sugars equivalent of
125.8 mg/g in 0.5% H2SO4. The minimum yield was 53 mg/g obtained in
3% H2SO4. The yield of bioethanol production by S. cerevisiae S288c
was (51.37 mg /g) was achieved at pH 5.5, temperature of 30⁰C and
inoculums size of 10% (v/v) after 72 hours of fermentation.
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‫إنتاج اإليثانول الحيوي من مخمفات البطاطس النشوية بواسطة‬
‫‪Saccharomyces cerevisiae‬‬
‫محمد جابر عبدالفضيل طه‪ -‬أحمد السيد خطاب‪ -‬حمدى السيد عمى‪-‬‬
‫محمد عوض محمود عبدالرحيم داود‬
‫قسم الكيمياء الحيوية الزراعية ‪ -‬كمية الزراعة‪ -‬جامعة األزهر‪.‬‬
‫يعتبر االيثانول الحيوى من أىم مصادر الطاقة الحيوية المتجددة الواعدة فى المستقبل‬
‫ولو فؤائد بيئية واقتصادية‪ ،‬و يمكن إنتاجو من مصادر مختمفة‪ .‬وقد اختيرت مخمفات البطاطس‬
‫النشوية كمصدر متجدد لمكربون إلنتاج اإليثانول ألنيا غير مكمفة نسبياً مقارنة بالمواد الخام‬
‫األخرى التي تعتبر مصادر غذائية‪ .‬وتجرى عمميات التحمل المائي باألحماض المخففة لتحويل‬
‫مخمفات البطاطس النشوية إلى سكرات قابمة لمتخمير (سكرات مختزلة) قبل عممية التخمر‬
‫إلنتاج اإليثانول‪ .‬في ىذه الدراسة تم تحسين التحمل المائي لمخمفات البطاطس النشوية ومعدالت‬
‫النمو لمخمائر لمحصول عمى أقصى قدر من إنتاج اإليثانول بواسطة ‪S. cerevisiae‬‬
‫‪ .S288c‬وكانت نسبة المواد النباتية إلى محمول الحمض ‪(11 :1‬و‪/‬ح) وأظيرت النتائج أن‬
‫حمض الكبريتيك تركيز ‪ ٪1,5‬و‪ ٪1‬و‪ ٪2‬و‪ ٪3‬عند ‪º121‬م لمدة ‪21‬دقيقة بواسطة‬
‫األوتوكالف كانت كافية لتحمل جميع النشا الموجود في مخمفات البطاطس النشوية‪ .‬وكان الحد‬
‫األقصى لكمية السكريات القابمة لمتخمير أو المختزلة ‪125.1‬ممجم‪/‬جم تم الحصول عمييا عند‬
‫تركيز الحمض ‪ .٪1,5‬وكان الحد األدنى من السكريات القابمة لمتخمير أو المختزلة ‪53‬ممجم‪/‬جم‬
‫التي تم الحصول عمييا عند تركيز ‪ .٪ 3‬و تم تحقيق أعمى إنتاج من اإليثانول الحيوي‬
‫بواسطة ‪ S. cerevisiae S288c‬وىو ( ‪ 51,33‬جرام ‪ 111 /‬جم) في درجة حموضة ‪،5.5‬‬
‫ودرجة ح اررة ‪º31‬م وحجم التمقيح ‪( ٪ 11‬و ‪ /‬و) بعد ‪ 32‬ساعة من التخمير‪.‬‬

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