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Waste Management 30 (2010) 18981902

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Ethanol production from potato peel waste (PPW)


D. Arapoglou a,1, Th. Varzakas b,*, A. Vlyssides c, C. Israilides a
a

Institute of Technology of Agricultural Products, National Agricultural Research Foundation, 1, Sof. Venizelou St., Lycovrissi, 14123 Athens, Greece
Technological Educational Institution of Kalamata, Department of Food Technology, Antikalamos, 24100 Kalamata, Greece
c
National Technical University of Athens, Department of Chemical Engineering, 9, Heroon Polytechniou St., Zographou 157 70, Greece
b

a r t i c l e

i n f o

Article history:
Received 9 July 2009
Accepted 7 April 2010
Available online 14 May 2010

a b s t r a c t
Considerable concern is caused by the problem of potato peel waste (PPW) to potato industries in Europe.
An integrated, environmentally-friendly solution is yet to be found and is currently undergoing investigation. Potato peel is a zero value waste produced by potato processing plants. However, bio-ethanol produced from potato wastes has a large potential market. If Federal Government regulations are adopted in
light of the Kyoto agreement, the mandatory blending of bio-ethanol with traditional gasoline in amounts
up to 10% will result in a demand for large quantities of bio-ethanol. PPW contain sufcient quantities of
starch, cellulose, hemicellulose and fermentable sugars to warrant use as an ethanol feedstock. In the
present study, a number of batches of PPW were hydrolyzed with various enzymes and/or acid, and fermented by Saccharomyces cerevisae var. bayanus to determine fermentability and ethanol production.
Enzymatic hydrolysis with a combination of three enzymes, released 18.5 g L 1 reducing sugar and produced 7.6 g L 1 of ethanol after fermentation. The results demonstrate that PPW, a by-product of the
potato industry features a high potential for ethanol production.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
With the inevitable depletion of the worlds energy supply,
there has been an increasing worldwide interest in alternative energy sources (Lin and Tanaka, 2006). In recent years, increasing research and development efforts have been directed towards the
commercial production of ethanol as the most promising biofuel
from renewable resources. In many European countries the use
of bio-ethanol as an alternative fuel or gasoline supplement in
amounts up to 15% is highly recommended (Mojovic et al.,
2006). If Federal Government regulations are adopted based on
the Kyoto agreement, the mandatory blending of bio-ethanol with
traditional gasoline in amounts up to 10% will result in a demand
for large quantities of bio-ethanol. Many countries have implemented, or are in the process of implementing, programs providing for the addition of ethanol to gasoline. Fuel ethanol production
has increased remarkably due to the global demand to reduce oil
importation, thereby contributing towards boosting rural economies and improving air quality. The world ethyl alcohol production has reached approximately 51,000 million liters, whereas
the USA and Brazil are the main producers. On average, 73% of

* Corresponding author. Tel.: +30 2721045281; fax: +30 2721045234.


E-mail addresses: dimarap@yahoo.com (D. Arapoglou), theovarzakas@yahoo.gr
(Th. Varzakas), avlys@tee.gr (A. Vlyssides).
1
Tel.: +30 2102845940; fax: +30 2102852521.
0956-053X/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.wasman.2010.04.017

the ethanol produced globally corresponds to fuel ethanol, 17%


to beverage ethanol and 10% to industrial ethanol (Sanchez and
Cardona, 2008).
The EU directive (2003/30/EC) for bio-ethanol requires member
states to establish legislation pertaining to the utilization of fuel
from renewable resources. In 2005, this utilization should cover
2% of the total fuel consumption. This quota is expected to increase
to 5.75% in 2010 and beyond (Berna, 1998). In the EU the annual
bio-ethanol production was 2155 million liters in 2008 (USDA,
2008).
Among the bioenergy crops used for fuel ethanol production,
sugarcane is the main feedstock utilized in tropical countries such
as Brazil and India. In North America and Europe, fuel ethanol is
mainly obtained from starchy materials, especially corn. Countries
with a signicant agricultural-based economy may apply the current technologies for fuel ethanol fermentation. It is estimated that
feedstock accounts for about 2055% of total production costs (Lin
and Tanaka, 2006; Del Campo et al., 2006), based on the total
estimated cost of USD 0.40/l of ethanol produced. Furthermore,
intensive research on the utilization of lignocellulosic biomass as
feedstock has been conducted over recent years. Any further
increase in ethanol production will necessarily involve the use of
feedstock rather than corn grain due to limited supplies. Feedstocks are typically grouped under the heading biomass and
include agricultural residues, wood, municipal solid waste and
dedicated energy crops (Kim and Dale, 2004; Stichnothe and
Azapagic, 2009). Biomass is seen as an interesting energy source

D. Arapoglou et al. / Waste Management 30 (2010) 18981902

for several reasons, the main reason being the contribution


provided by bioenergy to sustainable development (Sanchez and
Cardona, 2008). Resources are often available locally, and conversion into secondary energy carriers is feasible without high capital
investments (Lin and Tanaka, 2006).
The problem of the management of potato peel waste (PPW)
causes considerable concern to the potato industries in Europe,
thus implying the need to identify an integrated, environmentally-friendly solution. Potato peel is a zero value waste from potato processing plants.
While consumption of potatoes has decreased, processed products such as French fries, chips, and puree have experienced growing popularity (ZMP, 2000). Losses caused by potato peeling range
from 15% to 40% their amount depending on the procedure applied,
i.e. steam, abrasion or lye peeling (Scieber et al., 2001). Plants peel
the potatoes as part of the production of crisps, instant potatoes
and similar products. The produced waste is 90 kg per Mg of inuent potatoes and is apportioned to 50 kg of potato skins, 30 kg
starch and 10 kg inert material. The downstream processing in
the potato crisp industry is illustrated in the following general
owchart of Fig. 1 (Vlyssides et al., 2007).
The PPW contains sufcient quantities of starch, cellulose,
hemicellulose, lignin and fermentable sugars to warrant use as
an ethanol feedstock. Starch is a high yield feedstock for ethanol
production, but its hydrolysis is required to produce ethanol by
fermentation. Starch was traditionally hydrolyzed by acids, but
the specicity of the enzymes, their inherent mild reaction conditions and the absence of secondary reactions have led to the widespread use of amylases as catalysts in this process. Starch
processing is a technology utilizing enzymatic liquefaction and
saccharication, which produces a relatively clean glucose stream
that is fermented to ethanol by Saccharomyces yeasts (Gray et al.,

1899

2006). Enzymes afford numerous advantages compared to acidic


hydrolysis because they work under mild conditions, are biodegradable, improve yields, reduce energy, water consumption and
the amount of by-products (Israilides et al., 2008). The strategy
for the use of enzymes in the production of bio-ethanol from starch
includes two stages: liquefaction and saccharication. In liquefaction a-amylase, obtained either from thermoresistant bacteria
such as Bacillus licheniformis or from engineered strains of Escherichia coli or Bacillus subtilis are used to decrease viscosity in the
slurry or produce dextrins. In saccharication the enzymes use
dextrins to make glucose (Sanchez and Cardona, 2008).
In this paper we present a new PPW hydrolysis with a specic
combination of enzymes and/or hydrochloric acid, subsequently
fermented by Saccharomyces cerevisae var. bayanus to determine
fermentability and ethanol production. The novelty of the
approach lies in the application of the specic enzyme mix, which
includes a cellulolytic enzyme, aiming at the highest possible
fermentable sugar release for the production of ethanol.

2. Materials and methods


Samples of PPW were supplied to the Biotechnology Laboratory
of the National Agricultural Research Foundation (N.AG.RE.F.) in
Greece and were dried. Moisture was determined by oven drying
at 105 C to a constant weight. Total sugars and total carbohydrates were estimated by the colorimetric method of Dubois
et al. (1956). Protein was estimated by the Kjeldahl method by
multiplying residual nitrogen (N) by 6.25. Fat was determined by
the Soxhlet method (AOAC, 1995). Heavy metal analysis was performed by atomic absorption according to the standard methods
for examination of water and wastewater, (APHA, 1989). Starch

Fig. 1. Diagram of the potato crisps manufacturing and production of liquid wastes.

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D. Arapoglou et al. / Waste Management 30 (2010) 18981902

was quantied with the Kit. Cat. No. 10207748035, Behringer


Mannheim/R-Biopharm. Ethanol was measured with the Behringer Mannheim/R-Biopharm, Kit. Cat. No. 10 176 290 035. The
degree of polysaccharide degradation was estimated by quantifying the amount of reducing sugars formed during enzymatic or
acidic hydrolysis. Reducing sugars were determined as glucose
by using dinitrosalicylic acid (DNS) reagent at optical density
575 nm, by the method described by Miller (1959).
2.1. Microorganism
S. cerevisae var. bayanus from the Wine Institute (N.AG.RE.F.)
collection was used for the fermentation of hydrolyzed potato peel
waste (PPW), and was maintained on a malt agar slant. The agar
slant consisted of malt extract (3 g L 1), yeast extract (3 g L 1),
peptone (5 g L 1), agar (20 g L 1) and distilled water (up to 1 L).
For the inoculum, the culture was grown aerobically in 250 ml
asks in a shaking water bath at 32 C for 48 h. The liquid media
consisted of yeast extract (3 g L 1), peptone (3.5 g L 1), KH2PO4
(2 g L 1), MgSO47H2O (1 g L 1), (NH2)2SO4 (1 g L 1), glucose
(10 g L 1) and distilled water. Six percent of inoculum was used
for the fermentation of PPW.

2.3.2. PPW hydrolysis by enzymes


Two grams of dry potato peel waste (PPW) was mixed with
100 ml distilled water. The mixture was treated with enzymes in
two or three steps. The rst step, liquefaction, was carried out with
Ternamyl 120 L at 85 C and pH 6.0 for 1 h, or with Liquozyme Supra at 55 C, pH 5.5 and reaction time 20 h. The second step, saccharication, was carried out at 44 C, pH 4.6 for 2.5 h reaction
time with Viscozyme. An extra step with Celluclast to degrade cellulose was carried out at 50 C, pH 5 for 2 h reaction time. At the
end of each stage, the enzyme was deactivated with boiling in a
water bath at 95 C, pH 4.6 and for 10 min.
The enzyme combinations tested were the following:
a.
b.
c.
d.
e.

Liquozyme Supra (L) 1% (v/w) and Viscozyme (V) six FBGU.


Ternamyl 120 L (T) 0.24 KNU and Viscozyme (V) six FBGU.
Ternamyl 120 L (T) 0.48 KNU and Viscozyme (V) 12 FBGU.
Ternamyl 120 L (T) 2.4 KNU and Viscozyme (V) 12 FBGU.
Ternamyl 120 L (T) 0.24 KNU, Viscozyme (V) six FBGU, Celluclast (C) 0.5%.
f. Ternamyl 120 L (T) 0.24 KNU, Viscozyme (V) 12 FBGU, Celluclast (C) 1%.
g. Ternamyl 120 L (T) 0.24 KNU, Viscozyme (V) 24 FBGU, Celluclast (C) 2%.

2.2. Acidic hydrolysis


In the present study HCl was used to achieve acidic hydrolysis.
Hydrochloric acid is usually used for complete hydrolysis of plant
origin carbohydrates to simple reducing sugars, with no adverse effects on the material. To an 250 ml Erlenmeyer ask with fermentation trap, 40 g of PPW containing 85% moisture, 6 g dry matter
(15%) and 52.09% starch per dry weight, was added together with
0.5% NH4NO3, 0.1% peptone and 120 ml HCl 0.5 M. The mixture
was sterilized at 121 C for 15 min. During sterilisation the carbohydrates from potato peel were transformed into fermentable sugars due to acid hydrolysis. After sterilisation the pH was corrected
to 4.15 with NaOH (1 M).
2.3. Enzymatic hydrolysis
2.3.1. Enzymes
For the enzymatic hydrolysis of PPW the following enzyme
preparations from Novozymes A/S, Denmark, were used:
 Viscozyme L (V). Viscozyme is a cell wall degrading enzyme
complex from Aspergillus aculeatus. The activity of Viscozyme
L was 120 Fungal Beta-Glucanase Units (FBGU)/ml. One FBG is
the amount of enzyme required under standard conditions
(30 C, pH 5.0, reaction time 30 min) to degrade barley a-glucan
to reducing carbohydrates with a reduction power corresponding to 1 lmol glucose/min.
 Ternamyl 120 L (T). Ternamyl 120 L is a heat-stable amylase
from B. licheniformis. The enzyme activity was 120 KNU/g (kilo
novo units of a-amylase). KNU is the amount of enzyme
required to break down 5.26 g of starch per hour according to
Novozymes standard method for the determination of
a-amylase.
 Liquozyme Supra (L). Liquozyme Supra is a heat-stable a-amylase
from Bacillus lichneniformis. The enzyme activity was 200
KNU/g.
 Celluclast 1.5 L (C). Celluclast 1.5 L is a liquid cellulase preparation with an enzyme activity of 1500 NCU/g. One Novo Cellulase
Unit (NCU) is the amount of enzyme which, under standard
conditions, degrades carboxymethylcellulose to reducing carbohydrates with a reduction power corresponding to 1 lmol glucose per minute. It is produced by submerged fermentation of a
selected strain of Trichoderma reesei.

2.4. Ethanol fermentation of PPW hydrolyzates


Starch hydrolyzates obtained by both enzymatic and acidic
hydrolysis, were subjected to ethanol fermentation by a 48 h old
culture of Saccharomyces cereviciae var. bayanus under anaerobic
agitated conditions (pH 5.0, 32 C, 100 rpm) in a 250 ml Erlenmeyer ask with fermentation trap. The inoculum was 6% (v/v)
and fermentation was carried out for 2 days.
2.5. Statistical analysis
Samples were taken at random in triplicate runs. The results
were calculated as the average (means) of three separate measurements of each run. The means were compared with Students t-test
at a probability level of p = 0.05. The selection of Students t-test for
comparison of the means was chosen due to the fact that the distribution of t statistic is relatively stable for population that posses
not only a normal probability distribution but also a non-normal
mount-shaped one.
3. Results and discussion
Dry potato peel waste (PPW) composition is given in Table 1.
Soluble sugar, reducing sugar and starch are part of total carbohydrates. Lignin was not estimated. As shown, potato peel waste
(PPW) had a high starch content (52% d.w.) but the fermentable
reducing sugar was very low (0.6% d.w.). For this reason, any

Table 1
Chemical composition of potato peel waste (PPW).

Parameters

Dry weight (%)

Moisture %
Total carbohydrate
Total soluble sugar
Reducing sugar
Starch
Nitrogen
Protein (Ntot 6.25a)
Fat
Ash

85.06
68.7
1
0.61
52.14
1.3
8
2.6
6.34

Lignin was not estimated.

1901

D. Arapoglou et al. / Waste Management 30 (2010) 18981902

Fig. 2. Ethanol production (g L

) of soluble fermentable sugar from acidic hydrolysis of PPW.

fermentation of the raw material is not practical and an initial


hydrolysis (acidic or enzymatic) of carbohydrates is necessary.
3.1. Ethanol production from PPW
3.1.1. Acidic hydrolysis
After acidic hydrolysis the total amount of sugars was
19.37 g L 1 while the fermentable reducing sugar was 18.15 g L 1,
corresponding to 0.36 g released sugar per g of raw dry PPW. At
the end of fermentation total sugars were 4.34 g L 1 and fermentable reducing sugars 4.06 g L 1. Therefore, the reducing sugars consumed were 14.08 g L 1. The nal pH at the end of fermentation
was 3.89.
Fig. 2 shows the evolution of ethanol production (g L 1) during
PPW hydrolyzate fermentation. The maximum ethanol produced
was 6.97 g L 1 after 48 h fermentation, subsequently leveling off
to 60 h with a slight decrease thereafter. The product yield Y p/s
(g of product/g of sugar consumed) was 0.463 (or 46.3%). This corresponds to 92.6% of the max theoretical yield obtained when the
substrate is pure glucose (Bryan, 1990).
3.1.2. Enzymatic hydrolysis
The degree of hydrolysis of native starch of PPW depends on
factors such as substrate concentration, type and concentration
of the enzyme used, and the process conditions such as pH,
temperature, etc. Firstly, the ability of each enzyme to degrade

Table 2
Fermentable reducing sugars released after enzymatic hydrolysis of PPW with various
enzyme combinations, reducing sugar (g L 1) at the end of fermentation and
fermentable sugar during fermentation.
Enzyme combinations

a
b
c
d
e
f
g

L-1% and V-6U


T-0.24U and V-6U
T-0.48U and V-12U
T-2.4U and V-12U
T-0.24U and V-6U
and C-0.5%
T-0.24U and V-12U
and C-1%
T-0.24U and V-24U
and C-2%

PPW carbohydrates to fermentable reducing sugars was tested.


The use of Ternamyl released 4.65 g L 1, the Liquozyme
4.03 g L 1 and Viscozyme only 0.7 g L 1. Ternamyl and Liquozyme
are enzymes for starch liquefaction, and Viscozyme is used in saccharication. These results indicate that the saccharication stage
alone was inadequate and a preliminary liquefaction stage was required. For this reason, the use of enzyme combination was necessary for an effective hydrolysis of PPW.
Table 2 shows the reducing sugar released after enzymatic
hydrolysis, the remaining reducing sugar after fermentation by
S. cerevisae var. bayanus, as well as sugar consumed for fermentation. Due to lower release of reducing sugar and high reaction time
(20 h), the use of Liquozyme was rejected for starch liquefaction
and Ternamyl was selected with higher hydrolysate reducing sugar
and shorter reaction time (1 h).
The results indicate that higher enzyme concentration leads to
higher fermentable reducing sugar content. Obviously, the same
conversions could be achieved with lower enzyme concentration,
although requiring longer times. The longer exposure of the enzyme to high temperatures (85 C for Ternamyl), needed for gelatinization of the starch granules and to achieve a good susceptibility
to enzyme action could lead to slight enzyme deactivation (Mojovic et al., 2006).
On the other hand, treatment of PPW with Celluclast increased
the release of reducing sugars signicantly (p < 0.05), due to
the additional cellulose degradation. The increase of Celluclast

Table 3
Ethanol production (g L
fermentation.

Hydrolysate
reducing
sugar

Reducing
sugar after
fermentation

Consumed
sugar

14.06 0.67a
10.27 0.98b
16.82 1.01c
14.68 1.20a
16.99 1.17c

1.46 0.12
1.09 0.22
1.82 0.19
1.61 0.31
2.03 0.53

12.60 0.33
9.18 0.17
15.00 1.14
13.07 0.99
14.96 1.25

a
b
c
d
e

18.32 1.23d

2.11 0.28

16.21 1.54

18.48 0.65d

1.93 0.31

16.55 0.85

L: Liquozyme.
T: Ternamyl.
V: Viscozyme.
C: Celluclast.
a,b,c,d
Different superscripts are statistically signicant, p = 0.05.

), yield and % of maximum theoretical yield, during PPW

Enzyme combinations

Ethanol (g L

L-1% and V-6U


T-0.24U and V-6U
T-0.48U and V-12U
T-2.4U and V-12U
T-0.24U and V-6U and C0.5%
T-0.24U and V-12U and C1%
T-0.24U and V-24U and C2%

Y (p/s)

% Max theoretical
yield

5.86 0.15a
4.19 0.10b
7.00 0.06c
6.05 0.23a
6.89 0.47c

0.462
0.459
0.467
0.460
0.456

92.40
91.80
93.38
92.10
91.24

7.58 0.24c

0.463

92.54

7.50 0.28c

0.458

91.64

L: Liquozyme.
T: Ternamyl.
V: Viscozyme.
C: Celluclast.
a,b,c,
Different superscripts are statistically signicant, p = 0.05.

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D. Arapoglou et al. / Waste Management 30 (2010) 18981902

concentration from 1% to 2% did not lead to any signicant increase


of reducing sugar (p > 0.05). The enzyme combination with the
highest release of reducing sugar was (g) Ternamyl 0.24
KNU + Viscozyme 24 FBGU + Celluclast 2% with a rate of
18.48 g L 1 that corresponded to 0.92 g released sugar per g of
raw dry PPW. Using this combination, at the end of fermentation
the reducing sugars were 1.93 g L 1. Therefore, the sugars consumed were 16.55 g L 1 (Table 2). In all treatments the non-fermented sugar at the end of fermentation was very low. The
consumed sugar during fermentation was very high (up to 89%),
indicating the high ability of S. cerevisiae var. bayanus to bioconvert
reducing sugars.
Table 3 shows the ethanol production (g L 1), product yield Y
p/s (g of product/g of sugar consumed) and percentage of the maximum theoretical yield, with the use of various enzyme combinations. In all enzyme treatments, S. cerevisae produced high
quantities of ethanol (6.07.6 g L 1), with the exception of combination (b) resulting in lower ethanol production (4.2 g L 1). The
product yield Y p/s (g of product/g of sugar consumed) was about
0.46. In all cases the product yield corresponded up to 91% of the
max theoretical yield.
The results indicate that the critical parameters for ethanol production from PPW were the enzyme combination, the dose and the
residence time of hydrolysis. After liquication and saccharication, fermentation by S. cerevisae converted sugars to high yields
of ethanol.
4. Conclusions
It is evident that enzymatic hydrolysis of PPW liberates a higher
amount of fermentable reducing sugar compared to hydrochloric
acidic hydrolysis. The enzyme combination featuring the highest
release of reducing sugar, was (g) Ternamyl 0.24 KNU + Viscozyme
12 FBGU + Celluclast 1% with 18.48 g L 1. With this combination, at
the end of fermentation the reducing sugars were 1.93 g L 1.
Therefore, the sugars consumed were 16.55 g L 1. Ethanol production was 7.58 g L 1 and ethanol yield corresponded to 91.6% the
max theoretical yield.
The results demonstrated that PPW, a by-product of the potato
industry, could be efciently utilized for ethanol production with
simultaneous reduction of waste by-product. The production of
ethanol from PPW represents an alternative, readily manageable
option; the feasibility of implementing this technique will be further investigated by means of a larger scale technico-economical

study. Moreover, the availability of manufacturers to become involved in this project and to devote resources to its implementation will provide a signicant contribution.
Acknowledgements
The authors wish to thank Mrs. Malli Basiliki and Mrs.
Chaidemenaki Katerina for their valuable help in the analytical
work.
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