Applied Energy 140 (2015) 6574

Contents lists available at ScienceDirect

Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

A low-energy, cost-effective approach to fruit and citrus peel waste

processing for bioethanol production
In Seong Choi a, Yoon Gyo Lee a, Sarmir Kumar Khanal b, Bok Jae Park c, Hyeun-Jong Bae a,d,
a

Department of Wood Science and Landscape Architecture, Chonnam National University, Gwangju 500-757, Republic of Korea
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, United States
c
Division of Business and Commerce, Chonnam National University, Yeosu 550-749, Republic of Korea
d
Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
b

h i g h l i g h t s
! Simple bioprocess of bioethanol production from fruit wastes containing D-limonene.

! Two in-house enzymatic bioconversion rates were approximately 90%.

! Limonene recovery column (LRC) was designed for absorption of D-limonene.

! Ethanol production by immobilized yeast fermentation and LRC was 12-fold greater.

a r t i c l e

i n f o

Article history:
Received 22 March 2014
Received in revised form 17 November 2014
Accepted 29 November 2014
Available online 13 December 2014
Keywords:
Citrus peel waste
Bio ethanol
Enzymatic hydrolysis
D-Limonene extract
Continuous immobilized yeast fermentation

a b s t r a c t
Large quantities of fruit waste are generated from agricultural processes worldwide. This waste is often
simply dumped into landfills or the ocean. Fruit waste has high levels of sugars, including sucrose, glucose, and fructose, that can be fermented for bioethanol production. However, some fruit wastes, such as
citrus peel waste (CPW), contain compounds that can inhibit fermentation and should be removed for
efficient bioethanol production. We developed a novel approach for converting single-source CPW (i.e.,
orange, mandarin, grapefruit, lemon, or lime) or CPW in combination with other fruit waste (i.e., banana
peel, apple pomace, and pear waste) to produce bioethanol. Two in-house enzymes were produced from
Avicel and CPW and were tested with fruit waste at 1215% (w/v) solid loading. The rates of enzymatic
conversion of fruit waste to fermentable sugars were approximately 90% for all feedstocks after 48 h. We
also designed a D-limonene removal column (LRC) that successfully removed this inhibitor from the fruit
waste. When the LRC was coupled with an immobilized cell reactor (ICR), yeast fermentation resulted in
ethanol concentrations (14.429.5 g/L) and yields (90.293.1%) that were 12-fold greater than products
from ICR fermentation alone.
! 2014 Elsevier Ltd. All rights reserved.

1. Introduction
The world consumed approximately 89 million barrels of crude
oil per day in 2013. Consumption of liquid fuels (mainly petroleum) is expected to increase to 115 million barrels per day by
2040, which is a 63% overall increase in total liquid fuel consumed.
The consumption of liquid fuel by the transportation sector will
increase by 57% by 2040 [1]. The transportation sector is a source
of emissions of carbon dioxide (CO2) and other greenhouse gases

Corresponding author at: Department of Bioenergy Science and Technology,

Chonnam National University, Gwangju 500-757, Republic of Korea. Tel.: +82 62
530 2097; fax: +82 62 530 0029.
E-mail address: baehj@chonnam.ac.kr (H.-J. Bae).
http://dx.doi.org/10.1016/j.apenergy.2014.11.070
0306-2619/! 2014 Elsevier Ltd. All rights reserved.

(GHG) such as nitrogen oxide (NOx) and sulfur oxide (SOx). Biofuels
are and alternative energy source that reduce the production of
pollution gases [2]. The production of nonpetroleum liquid fuels,
such as biofuels, from food crops is not sustainable due to competition for materials and high production costs. Therefore, cheap and
abundant nonfood materials are required as alternative biomass
feedstocks (e.g., agricultural byproducts, woody biomass, or energy
crops) and processes must be developed that can efficiently and
economically convert these types of lignocellulosic and cellulosic
biomass into biofuels, such as bioethanol [3].
Fruit waste is generated in large quantities from the processing
of agricultural products. Examples of such waste include citrus,
banana, apple, and pear residues remaining after industrial processing. Citrus, which includes oranges, grapefruits, lemons, limes,

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I.S. Choi et al. / Applied Energy 140 (2015) 6574

mandarins, are the most abundant crops in the world. Over 115
million tons of citrus fruits are produced annually, and about 30
million tons are processed industrially for juice production. After
industrial processing, citrus peel waste (CPW) accounts for almost
50% of the wet fruit mass. The annual production of bananas,
apples, and pears are approximately 107.1, 75.5, and 24.0 million
tons, respectively, and 2540% of this mass remains as waste after
processing (Fig. 1A) [4]. Fruit waste serves as cattle feed, but
because of its low protein content, it is not a high-value feedstock,
and much of it is dumped into landfills or disposed of in the ocean.
Because fruit waste is rich in sugars and other nutrients, these
forms of disposal may cause environmental problems. Disposal of
waste is also becoming increasingly expensive. For example, European Union (EU) landfill directives have caused landfill gate fees to
increase in some cases because of land limitations and transport
and labor costs [5]. In America, the annual cost of apple pomace
disposal alone is $10 million USD [6]. Fruit waste is rich in fermentable soluble sugars such as glucose, fructose, and sucrose along
with structural cellulose and hemicellulose. These chemical constituents, along with the fact that fruit waste is in abundant supply,
suggest that fruit waste may be an excellent source of waste biomass for ethanol production.
However, among the variety of fruit wastes available, CPW
requires additional processing before bioethanol production. This

is because although CPW is rich in various soluble and insoluble

sugars, making it an ideal feedstock, it also contains a strong
microbial inhibitor referred to as D-limonene. The production of
D-limonene from citrus peel is economically viable, as this byproduct has high added value as a flavoring agent and for various
applications in the chemical industry. Thus, removing and recovering D-limonene prior to the yeast fermentation process serves two
purposes: high-value utilization and enhanced fermentation of
CPW-derived sugars. The efficient removal of D-limonene from
CPW requires a pretreatment step. Most pretreatment methods
are based on thermochemical or thermophysical processes such
as milling or steam explosion as shown in Fig. 1B and Table 1
[714]. A major disadvantage of these methods is the elevated
temperature and prolonged extraction time, which can cause
chemical modification of the volatile molecules, including D-limonene,
as well as loss of sugars for ethanol production. We developed a
new technique that uses raw cotton and activated carbon to
remove and recover D-limonene, and requireds with less energy.
Sorbents should have high oleophilic and hydrophobic properties,
and can be classified into three groups based on the material
source (natural materials, treated cellulose, or petrochemical polymers). The most commonly used polymers are petrochemical polymers such as polypropylene, polyethylene, and polyurethane.
However, these polymers are non-biodegradable materials and

Fig. 1. Citrus and fruit production and schematic representation of bioethanol production processes. (A) Annual production of citrus and major fruit worldwide, (B)
traditional processes for citrus peel bioethanol production, and (C) schema of the study process. In most cases, steam explosion pretreatment is convention process to remove
the fermentation inhibitor D-limonene. Citrus peel was hydrolyzed by commercial enzymes, including pectinase, cellulase, and b-glucosidase.

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I.S. Choi et al. / Applied Energy 140 (2015) 6574

Table 1
Citrus waste as substrate for bioethanol production.
Substrate
Orange peel
Orange peel
Orange peel
Orange peel
Orange peel
Mandarin peel
Mandarin peel
Lemon peel
a
b
c
d

Pretreatment
Milling
Milling
Steam explosion
Steam explosion
Acidic steam explosion
Steam explosion
Popping
Steam explosion

Enzymesa
Pectinase,
Pectinase,
Pectinase,
Pectinase,
Pectinase,
Pectinase,
Pectinase,
Pectinase,

Fermentation process
cellulase,
cellulase,
cellulase,
cellulase,
cellulase,
cellulase,
xylanase,
cellulase,

glucosidase
glucosidase
glucosidase
glucosidase
glucosidase
glucosidase
glucosidase
glucosidase

HF
HF
SSF
SSF
SSF
SSF
SHEFb
SSF

Microorganism
S. cerevisiae
Escherichia coli KO11
S. cerevisiae
Kluyveromyces marxianus
S. cerevisiae
S. cerevisiae
S. cerevisiae
S. cerevisiae

Ethanol production
c

4.7
2.76c
3.96c
3.45c
2.7c
59.3d
46.2d
67.8d

References
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]

Commercial enzymes used for hydrolysis.

SHEF, separate hydrolysis and fermentation with vacuum evaporation.
Ethanol yields presented in%, w/v.
Ethanol concentration expressed in g ethanol per g of 1000 kg of fresh substrate.

can become environmental pollutants. Raw cotton is a natural

material that hydrophobic, with a high sorption capacity, and is
easily biodegradable [15]. Activated carbon possesses a high
degree of micro-porosity for absorption and is commonly used
for water treatment, detoxification, and separation of components
in flow systems.
In the enzymatic hydrolysis phase, cellulolytic, xylanolytic, and
pectinolytic enzymes are often used to degrade plant cell walls and
catalyze the breakdown of complex carbohydrates into their
monosaccharide components (i.e., saccharification) [12,13]. Ethanol production from CPW has largely been conducted using commercial enzymes; thus, the cost of cellulosic ethanol is very high.
The cost can be drastically reduced if in-house-produced enzymes
are used for saccharification [16]. Trichoderma and Aspergillus are
among the most common microorganisms that produce abundant
cellulolytic, xylanolytic, and pectinolytic enzymes. Trichoderma
species have been studied for their cellulolytic enzymes content
[17]. In addition, Aspergillus species often have xylanolytic and pectinolytic enzymes [18]. Both fungal species are considered extracellular producers of cell wall-degrading enzymes that have
potential for important industrial applications.
Following completion of enzymatic hydrolysis, fermentation is
necessary for bioethanol production. Generally, ethanol production
may occur through separate hydrolysis and fermentation (SHF)
processes, or simultaneous saccharification and fermentation
(SSF). Continuous fermentation is also considered an efficient fermentation process, because it has many advantages, including
the ability to separate immobilized yeast from the ethanol product,
thus allowing the immobilized yeast to be reused for further fermentation. In addition, immobilizing the yeast cell wall confers
higher ethanol tolerance and cell concentrations, shorter fermentation time, enhanced fermentation productivity, and lower costs of
recovery and recycling [19].
Considering the above-mentioned limitations of conventional
processes, we explored the possibility of directly converting fruit
waste to ethanol without pretreatment (Fig. 1B and C). This
involved developing an efficient enzymatic hydrolytic process, as
well as an effective, low-cost strategy to remove the fermentation
inhibitor D-limonene. The utility of this approach was examined by
evaluating ethanol production efficiency during continuous fermentation with immobilized yeast cells. Furthermore, because
feedstock flexibility is important for successful commercial ethanol
production, the feasibility of using CPW alone or in combination
with other fruit waste was also examined.
2. Materials and methods
2.1. Raw materials
Citrus (orange, mandarin, grapefruit, lemon, and lime), apple,
banana, and pear were obtained from a local market (Homeplus,

Gwangju, Korea). Citrus, apple, and pear waste was collected after
juice extraction (Hurom, Seoul, Korea). Banana waste was
removed, lyophilized ("50 "C), and stored at "20 "C. CPW and fruit
wastes, individually or mixed in equal ratios, waste were used for
hydrolysis and fermentation.
2.2. Chemical composition
The content of soluble sugar was analyzed by high performance
liquid chromatography (HPLC) with a refractive index detector
(2414, Waters, USA), REZEX RPM (Phenomenex, USA) column
(300 # 7.8 mm) at 85 "C and eluted with deionized water at a flow
rate of 0.6 mL per min. Insoluble solids were analyzed for neutral
sugar content using gas chromatography (GC) [20,21]. Samples
were hydrolyzed with 72% sulfuric acid for 45 min at room temperature and diluted with distilled water to 4% sulfuric acid, followed
by autoclaving for 1 h at 121 "C. The neutral sugar composition was
measured with alditol acetates containing myo-inositol as an internal standard. Gas chromatography (GC-2010, Shimadzu, Japan)
was used, and the analysis conditions, using a DB-225 capillary column (30 m # 0.25 mm i.d., 0.25 lm film thickness, J&W) operated
with He, injector temperature of 220 "C, flame ionization detector
(FID) at 250 "C, and oven temperature programming, were 100 "C
for 1.5 min and 5 "C/min to 220 "C.
The D-limonene content was determined according to a previous study [13]. Briefly, CPW was homogenized in 10 mL hexane,
which had a known amount of camphor as an internal standard.
After treatment for 3 h, 5 mL of supernatant was transferred to a
test tube. A quantity of 0.2 mL potassium hydroxide (2 N) was
added to methanol and mixed for 1 min. After the addition of
1 mL distilled water, the samples were shaken and centrifuged
for 5 min at 3000 rpm. The hexane phase was measured by GC
(CP-9100, Chrompack), using a CP-Sil 5 CB fused silica capillary column (25 m # 0.32 mm i.d., 1.2 lm film thickness, Chrompack)
operated with He, injector temperature 280 "C, FID at 280 "C, and
oven temperature programming at 110 "C for 5 min and 20 "C/
min to 220 "C, which was then held constant for 10 min.
2.3. In-house enzyme production
Aspergillus citrisporus (KCCM 11449) was obtained from the Korean Culture Center of Microorganisms (KCCM), and Trichoderma
longibrachiatum (KCTC 6507) was purchased from the Korean Collection for Type Cultures (KCTC). The lyophilized fungi were revitalized on potato dextrose broth with 1.2% (w/v) agar (PDA) and
incubated for spore production for 7 days at 25oC. One hundred
of potato dextrose broth (PDB) was sterilized in 500 mL Erlenmeyer flasks.
Two types of carbon sources were used to produce extracellular
enzymes for fruit waste hydrolysis. The medium contained either
20 g/L MP or Avicel as the carbon source. The other components

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I.S. Choi et al. / Applied Energy 140 (2015) 6574

were similar for both media (in g/L): 40, peptone; 24, KH2PO4; 5,
(NH4)2SO4; 4.7, K2C4H4O6$4H2O; 2, urea; 1.2, MgSO4$7H2O and (in
mg/L) 10; ZnSO4$7H2O, 9.3; MnSO4$7H2O, 8.7; CuSO4$7H2O with
1 mL Tween 80. The pH was adjusted to 5.0, using hydrochloric
acid. The medium was sterilized at 121 "C for 15 min. Cultures
were conducted in a 10 L fermenter (Fermentec, Korea) equipped
with a 7 L working volume for 7 d. The culture broth was centrifuged and the supernatant was stored at 4 "C.

2.5. D-Limonene recovery column design

The D-limonene removal column (LRC) is a tubular apparatus,
consisting of an internal diameter (ID) of 1.5 cm and 7.0 cm length.
LRC was packed with raw cotton (100300 mg) and activated-carbon (02 g) to optimize limonene removal. LRC was connected to
the fermentation reactor for D-limonene removal and recovery
from the hydrolysate prior to fermentation. The fermentation process was conducted with- or without LRC on a fermentation reactor. After fermentation, D-limonene was recovered from LRC using
hexane, and the recovery rate was determined using GC as
described in 2.2.

2.4. In-house enzyme activity and hydrolysis

Two enzymes, produced in-house from A. citrisporus (In-house
enzyme A [HEA]) and T. longibrachiatum (In-house enzyme B
[HEB]), were evaluated. The protein concentration was measured
using the Lowry method, with bovine serum albumin (BSA) as a
protein standard [22]. Enzyme activities were assayed with a specific substrate solution consisting of 50 mM citrate phosphate buffer, pH 4.8 (at 45 "C for 30 min), and appropriately diluted enzyme
concentrations. Endoglucanase (CMCase) and exoglucanase (Avicelase) activities were measured with 1% carboxymethylcellulose
(CMC, Sigma) and microcrystalline cellulose (Avicel, Sigma) as
the substrates, respectively. Xylanase activity was measured by
the same procedure described for endo- and exo-glucanase, but
with beechwood xylan (Sigma) as the substrate. Pectinase activity
was measured with a 0.5% polygalacturonic acid (Sigma) in 50 mM
citrate phosphate buffer (pH 4.8) at 45 "C for 5 min. Reducing sugars were quantified with dinitrosalicylic acid (DNS) at an absorbance of 540 nm [23]. One unit of activity was defined as the
amount of enzyme required to release one lmol of glucose, xylose,
or galacturonic acid per min. Specific activities were expressed as
enzyme units per milligram of protein.
HEA or HEB enzymes were added to fruit waste at concentrations of 1216 and 1025 mg protein/g fruit waste, respectively.
Enzymatic hydrolysis was performed on 1% matter (w/v) with citrate phosphate buffer (pH 4.8) at 180 rpm for 48 h at 45 "C. Optimization of enzyme loading volume and the influence of biomass
hydrolysis time during enzymatic hydrolysis were measured using
HPLC as described in 2.2.

2.6. Continuous immobilized yeast fermentation

Saccharomyces cerevisiae KCTC 7906 was obtained from the
KCTC and activated in 4 mL yeast peptone dextrose media (YPD).
The yeast inoculum was placed in a 500 mL Erlenmeyer flask containing 100 mL autoclaved YPD media for 24 h at 30 "C.
To prepare for immobilization, 100 mL S. cerevisiae cells were
harvested at the exponential growth phase and mixed with 2%
sodium alginate solution prepared by dissolving 8 g sodium alginate in 300 mL deionized water [24]. Using a syringe, the alginate
drops were deposited in a 0.1 M CaCl2 solution to produce beads.
The beads were stored after washing with deionized water to
remove any remnant CaCl2. The 3.8 mm beads were uniformly
packed and stored in deionized water at 4 "C.
The immobilized cell reactor (ICR) was used in continuous fermentation of the CPW hydrolysate. ICR consists of a tubular column, constructed with a 2.1 cm ID and 25 cm length. About 70%
of the column was packed with immobilized yeast cells. The medium was fed into the reactor from the feed stock, and a peristaltic
pump (EP-1 Econopump, Biorad) was used to transfer the feed
medium. The volumes of the reactor before and after immobilized
yeast cell packing were 80 and 42 mL, respectively. The fresh feed
was pumped in an up-flow manner and the total sugar and ethanol
concentrations were monitored during fermentation. Prior to being
fed into reactor, the pH of the CPW hydrolysates was adjusted to a

Table 2
Chemical compositions of fruit waste.
(% Dry matter)

Rhamnose

Arabinose

Xylose

Mannose

Galactose

Sucrose

Glucose

Fructose

FSa

Total

OP
MP
GP
LeP
LiP
AP
BP
PP
MixP
TFW

2.1 0.0
2.9 0.1
3.4 0.0
2.1 0.1
2.5 0.2
1.7 0.1
0.6 0.1
1.3 0.1
2.7 0.1
2.0 0.1

5.6 0.2
3.3 0.1
4.8 0.2
5.2 0.3
8.5 0.4
5.5 0.1
4.4 0.3
6.0 0.3
5.6 0.2
5.4 0.4

2.2 0.0
2.4 0.1
2.3 0.1
2.6 0.2
2.5 0.1
6.2 0.3
5.6 0.4
20.2 0.9
2.4 0.2
5.5 0.6

2.4 0.1
2.3 0.1
2.2 0.0
2.1 0.1
2.0 0.1
2.8 0.1
3.6 0.1
2.4 0.0
2.2 0.0
2.5 0.1

2.7 0.1
3.9 0.1
3.5 0.2
4.6 0.1
4.3 0.1
4.2 0.3
2.8 0.1
4.5 0.3
3.8 0.0
3.8 0.2

5.6 0.2
7.4 0.2
1.4 0.1
ND
ND
9.2 0.1
ND
1.9 0.1
2.9 0.1
3.2 0.3

35.5 0.5
39.4 1.1
30.6 0.8
27.9 0.4
22.5 1.2
25.2 2.8
30.1 0.8
21.1 0.6
32.0 0.8
29.0 1.7

12.1 0.4
10.3 0.8
8.2 0.3
3.3 0.1
0.7 0.0
24.7 0.3
15.2 0.7
14.1 0.5
6.8 0.3
11.1 0.9

53.2 0.4
57.1 0.6
43.2 0.7
31.2 0.4
23.2 1.0
59.1 1.8
45.3 0.3
37.1 0.8
41.7 1.1
43.4 1.9

68.2 0.5
71.9 0.9
59.4 0.8
47.8 0.3
43.0 0.6
79.5 1.5
71.5 0.6
62.3 0.7
58.4 0.8
62.5 1.7

Abbreviations used: OP, orange peel; MP, mandarin peel; GP, grapefruit peel; LeP, lemon peel; LiP, lime peel; AP, apple pomace; BP, banana peel; PP, pear peel; MixP, mixed
citrus peel; TFW, mixed total fruit wastes; ND, not detected.
Values represent the average of three replicates.
a
FS: Fermentable sugars are the sum of sucrose, glucose, and fructose, which are fermented by S. cerevisiae.

Table 3
Comparison of specific activities for the in-house enzymes used in the study.

In-house enzyme A (HEA)

In-house enzyme B (HEB)

Endoglucanase (U/mg protein)

Exoglucanase (U/mg protein)

Xylanase (U/mg protein)

Pectinase (U/mg protein)

8.41 0.11
13.22 1.21

0.18 0.01
1.26 0.17

170.95 1.81
4.34 0.52

17.90 0.43
1.11 0.21

69

Abbreviations used: OP, orange peel; MP, mandarin peel; GP, grapefruit peel; LeP, lemon peel; LiP, lime peel; MixP, mixed citrus peel; TFW, mixed total fruit wastes; HEA, in-house enzyme A; HEB, in-house enzyme B.

LiP
LeP

71.6 2.2
85.7 2.1
89.0 1.2
89.1 0.9
89.4 1.7
73.0 1.0
79.6 1.1
84.7 1.9
91.1 2.0
91.2 2.1

TFW
MixP

73.1 1.7
84.1 1.5
87.8 2.2
90.0 2.0
90.4 1.7
72.5 1.6
80.1 1.5
82.7 1.7
90.1 1.0
90.3 1.9

GP
MP

73.1 1.2
82.4 2.9
86.1 1.6
90.8 1.2
90.7 1.5
70.4 2.2
83.4 3.5
85.7 2.1
90.2 1.4
90.5 2.0

OP
LiP

70.6 1.2
81.1 0.8
88.7 1.2
88.9 1.7
88.9 2.0
70.1 1.9
82.1 2.0
89.0 2.1
89.1 1.8
89.1 2.1

LeP
TFW

69.1 1.7
72.8 1.6
79.8 2.1
84.1 1.8
85.8 3.5
66.8 1.5
75.4 1.2
77.1 3.3
76.2 2.2
83.4 1.1

MixP
GP
MP

64.0 0.9
71.6 1.5
73.6 3.1
78.7 1.6
80.1 1.2

OP

65.8 1.2
73.1 0.9
76.1 1.8
81.2 2.2
82.4 3.3

Group A

16 mg HEA /g fruit waste

Group B
Group A

3.2.1. In-house enzyme activity and effective loading volumes for

hydrolysis
Between the two in-house enzymes evaluated (Table 3), HEA
exhibited the highest level of xylanase activity. Its pectinase
activity was moderate, and both exoglucanase and endoglucanase activities were observed. The activity of xylanase and pectinase were lower for HEB, compared to that of HEA, but
endoglucanase and exoglucanase activities were higher or HEB.
Interestingly, HEA was produced using CPW as the carbon source,
and it showed high xylanase activity. This may have occurred
because hemicellulose forms a large component of the polysaccharides in CPW [13,26], and xylanase produced monosaccharides by CPW hydrolysis for fungal survival. Microorganisms
produce the appropriate complex enzymes for hydrolysis during
growth on a given substrate. The presence of hemicellulosederived saccharides in CPW is thought to be important for HEA
induction. Based on the above mentioned HEA and HEB activities,
we designed a synergistic cooperation between cellulolytic,
xylanolytic and pectinolytic enzyme mixtures to hydrolysis. To
determine the amount of enzyme necessary for fruit waste
hydrolysis, different enzyme volumes were loaded onto OP, MP,
GP, LeP, LiP, MixP, and TFW substrates. Data for the hydrolysis
of various fruit wastes by HEA and HEB are shown in Table 4.

12 mg HEA/g fruit waste

The current cost of pretreatment and enzymes for biomass

hydrolysis are major obstacles to large-scale ethanol production
[25]. The cost of cellulase is estimated at a minimum of $10/kg
protein [16]. Accordingly, it is necessary to reduce the cost and
amount of enzymes required for biomass hydrolysis to industrialize the process. Here, we produced a suitable enzyme complex for
fruit waste hydrolysis using CPW or Avicel as the carbon source.
In addition, we also report on the efficacy of the in-house enzyme
activities and fruit waste hydrolysis.

Mg HEB/g fruit waste

3.2. In-house enzyme production and fruit waste hydrolysis

Conversion rate (%)

The carbohydrate composition of the various fruit wastes differed as shown in Table 2. The total carbohydrate contents of the
fruit wastes were separated into soluble sugars, which dissolve
easily in water, and insoluble sugars (cellulose and hemicellulose) in the cell walls. Although arabinose and xylose were present, they appeared in low concentrations in the fruit waste. We
mainly focused on fermentable sugars (FS), namely, glucose, fructose, and sucrose. All fruit wastes presented were high in FS content (Table 2). Sucrose and fructose were present as soluble free
sugars, whereas, glucose was part of the fruit waste structural
components and present as a free sugar. FS contents in the various fruit wastes ranged from 23.2% to 59.1%. Orange peel (OP),
mandarin peel (MP), grapefruit peel (GP), apple pomace (AP),
and banana peel (BP) waste contained 53.2%, 57.1%, 43.2%,
59.1%, and 45.3% FS, respectively. Lemon peel (LeP), lime peel
(LiP), and pear pomace (PP) showed moderate FS levels of
31.2%, 23.2%, and 37.1%, respectively. FS contents in the CPW
mixture (MixP) and CPW, in combination with other fruit waste
(TFW), were 41.7% and 43.4%, respectively.

Table 4
Conversion rates for various types of citrus peel waste (CPW), alone or in combination with other fruit wastes after treatment with in-house enzymes (HEA and HEB) at different loading volumes.

3.1. Fruit waste composition

63.2 1.8
72.1 1.2
75.4 3.1
80.4 2.2
81.2 2.6

Group B

3. Results and discussion

0
10
15
20
25

pH of 4.8 by the addition of CaCO3. The flow rate of feed in the

packed-bed reactor column was 0.08 mL/min. The ICR was maintained in an incubator at 30 "C, and samples were withdrawn
aseptically from the bioreactor periodically during a 10-day period to analyze sugar and ethanol concentrations.

70.9 1.0
84.1 2.0
88.7 1.6
88.4 1.6
88.4 2.1

I.S. Choi et al. / Applied Energy 140 (2015) 6574

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I.S. Choi et al. / Applied Energy 140 (2015) 6574

Fig. 2. Waste-to-FS conversion rates as influenced by substrate loadings (%, w/w).

Table 5
Influence of enzymatic hydrolysis time on various kinds of citrus and mixed fruit waste.
Time (h)

12

15

18

21

24

48

Group A (12%)

OP
MP
GP
MixP
TFW

53.0 2.3
51.8 1.9
51.8 2.5
51.8 1.5
52.1 2.1

64.0 2.0
66.3 3.0
65.3 3.1
65.0 2.4
67.2 2.0

76.2 2.7
78.8 2.1
75.6 2.9
76.3 2.3
79.1 2.6

84.8 3.1
85.7 2.7
85.7 2.1
85.7 2.4
87.8 2.1

86.2 1.3
87.4 3.1
87.4 3.5
87.4 2.7
89.2 2.3

87.3 1.8
88.5 2.0
88.5 2.5
88.5 3.0
90.1 1.8

88.8 2.0
89.3 3.3
89.3 2.5
89.3 3.2
90.3 2.7

89.5 2.2
89.7 2.6
89.7 3.0
89.7 2.8
90.8 2.1

90.2 1.7
90.8 2.1
90.1 2.3
90.0 1.8
91.4 2.1

Group B (15%)

LeP
LiP

45.8 2.1
47.2 2.9

61.2 2.6
64.3 1.9

72.4 1.9
73.8 1.9

80.1 2.6
79.4 3.0

85.3 1.9
86.7 2.7

87.0 2.2
87.8 1.9

87.8 1.7
88.0 1.9

89.3 2.6
88.0 2.4

89.0 1.7
88.7 2.0

Abbreviations used: OP, orange peel; MP, mandarin peel; GP, grapefruit peel; LeP, lemon peel; LiP, lime peel; MixP, mixed citrus peel; TFW, mixed total fruit wastes.
Group A and B concentrations were 12% and 15% (w/w) solid loading, respectively.

Table 6
Summary of ethanol production from LRCICR system.

OP
MP
GP
MixP
TFW
LeP
LiP

Fermentable sugar content (g/L)

Enzymatic hydrolysate (g/L, %a)

Ethanol concentration (g/L)

Ethanol yield (%)

Productivity (g/L/h)

63.8
68.5
51.8
50.0
47.4
46.8
34.8

57.5/90.2%
62.2/90.8%
46.7/90.1%
44.4/90.0%
43.3/91.4%
42.1/89.0%
31.0/88.7%

27.1
29.5
21.6
20.4
20.3
19.6
14.4

92.4
93.1
90.7
90.2
91.8
91.1
90.8

3.01
3.28
2.40
2.27
2.26
2.18
1.60

Ethanol yield was calculated based on the fermentable sugars obtained from the hydrolysis of fruit waste.
Theoretical ethanol yield was assumed to be 0.51 g/g sugar.
a
Enzymatic hydrolysis efficiency.

Although effects on fruit waste hydrolysis rates were speciesdependent, the relatively low loading of HEA supplemented with
HEB achieved a high overall hydrolysis rate. An increase in FS concentrations from OP, MP, GP, MixP, and TFW (group A) was
observed when HEA levels were increased from 12 to 16 mg HEA
with 20 mg HEB per g fruit waste. The FS from LeP and LiP (group
B) increased at lower enzyme loadings (HEA 12 and HEB 15 mg/g
fruit waste) compared to loadings required in group A. The different chemical components of fruit waste may lead to differences in
enzymatic hydrolysis processes. However, FS concentration was
not increased significantly, even added more enzymes to group A
and B. This may have occurred because the hydrolysis of hemicellulose and pectin increases the surface area of the fruit waste and,

therefore, increasing accessibility and probability that the cellulose

will become hydrolyzed [27,28]. A combination of 16 mg HEA and
20 mg HEB, or 12 mg HEA and 15 mg HEB per g fruit waste, was
used in all further experiments for group A or B, respectively. In
this study, treatment with HEA invertase resulted in a decrease
in sucrose levels (through hydrolysis) and corresponding increases
in monomers fructose and glucose (Supplementary Fig. S1). The
hydrolysis of sucrose can be an issue in continuous bioethanol production. This is because S. cerevisiae shows preferential consumption of glucose and fructose over sucrose during fermentation,
and, as a result, sucrose is only consumed when the former two
substrates are exhausted. These differences in the kinetics of sugar
consumption may limit bioethanol production from fruit waste.

I.S. Choi et al. / Applied Energy 140 (2015) 6574

71

Fig. 3. Limonene removal and recovery. (A) The sorbent column was filled with raw cotton and activated carbon. (B) Citrus peel and mixed fruit waste contained different
D-limonene concentrations. (C) D-limonene from orange peel (black arrow) was detected by gas chromatography, before and after recovery, and (D) D-limonene was recovered
after fermentation.

According to Ghorbani et al. [29], to increase fermentation efficiency and avoid limitations, sucrose must be hydrolyzed to glucose and fructose via sucrose hydrolysis enzymes.
3.2.2. Influences of fruit waste concentration and time on enzymatic
hydrolysis
Based on the enzyme loading results in Table 4, we evaluated
the effects of varying fruit waste concentrations on the enzymatic
conversion of waste to FS. The conversion rates were calculated
based on the total FS of fruit waste. Fruit waste solid loadings varied from 3% to 18% (Fig. 2). For group A (OP, MP, GP, MixP, and
TFW) and group B (LeP and LiP), conversion rates decreased only
slightly as substrate loadings increased from 3% to 12% and 3% to
15%, respectively. After this point, further increases in substrate
loading significantly decreased conversion rates in group A
(>12%) and group B (>15%). Based on these results, all further
experiments used solid waste loadings of 12% for group A wastes,
and 15% for group B wastes. In addition to the influence of substrate loading, we examined the influence of hydrolysis time on
waste-to-FS conversion (Table 5). FS conversion rates were high
within the first 3 h, and considerable conversion of CPW to FS
was achieved within 9 h of hydrolysis. This kinetic behavior is in
agreement with our previous work [13], which showed rapid

CPW hydrolysis within the first hours of the reaction, followed

by a significant decrease. An FS conversion rate of approximately
85% was achieved within the first 12 h for group A. However, group
B required 15 h to reach a similar level of conversion. Conversion
rates did not increase significantly after 12 and 15 h in groups A
and B, respectively. From an economic perspective, these results
are favorable given that they permit a high degree of conversion,
which is necessary to maximize yield of ethanol during fermentation. Moreover, the overall hydrolysis time required was relatively
short compared to previous studies examining ethanol production
from lignocellulosic biomass [19,27,30].
3.3. D-limonene recovery and continuous immobilized yeast
fermentation

3.3.1. Development of a D-limonene adsorbent column

Citrus contains D-limonene, a terpenoid essential oil that inhibits yeast fermentation. In conventional processing, D-limonene is
removed and recovered using energy-intensive methods, such as
steam explosion. In contrast, conventional pretreatment method
has a major disadvantage, in that carbohydrate content of the feedstock may decrease to as low as 10% after pretreatment due to

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I.S. Choi et al. / Applied Energy 140 (2015) 6574

Fig. 4. Comparisons of fermentable sugar conversion and ethanol concentrations in ICR vs. LRCICR fermentation. (A) Initial FS concentrations in OP, (B) MixP, and (C) TFW
were 57.5, 44.4, and 47.4 g/L, respectively. (D) The glucose-to-ethanol conversion rates obtained after 10 days of fermentation. Black solid lines indicate the amount of FS from
ICR (
) or LRCICR (
) fermentation, and gray solid lines indicate the amount of ethanol produced from ICR (
) or LRCICR (
) fermentation.

losses resulting from the Maillard reaction, caramelization, and

oxidation [13,3133]. With the aim of developing a more costeffective, low-energy solution to this issue, we devised an LRC
made of raw cotton and activated carbon, and we evaluated its
removal efficiency through gas chromatography (GC).
To evaluate the effects of column packing on D-limonene
removal rate, various weights of raw cotton and activated carbon
were used to construct the LRCs. Decreasing D-limonene concentrations in the filtrate were observed when the raw cotton weight
was increased from 100 to 300 mg per column, as well as when the
quantity of activated carbon was increased from 0 to 1.5 g. However, further improvements in D-limonene adsorption were not
observed when using >2.0 g activated carbon. Thus LRCs containing
300 mg raw cotton and 1.5 g activated carbon (as shown in Fig. 3A)
were used in all further experiments. Analysis of the fresh CPW
showed contents of 0.3211.858% (w/w) D-limonene (Fig. 3B);
however, after passing through the LRC, D-limonene was undetectable. This result represented an improvement in D-limonene
removal compared to the conventional method used in previous
studies. In previous studies, inhibition of fermentation processes
was observed at concentrations greater than or equal to 0.1% (v/
w) [9]. Grohmann et al. [7] have shown inhibitory minimum concentrations, between 0.05% and 0.15% (v/w), that can affect the fermentation process. Moreover, about 90% of the D-limonene was
recovered after a 10-day fermentation period with a 0.08 mL/min
flow rate (Fig. 3C and D). These removal rates could be due to
the fact that D-limonene concentration and viscosity are low in
the hydrolysate. Because oil penetration rate into the internal surface of sorbents is inversely proportional to oil viscosity and concentration [34], adsorption should be high in the pores of the
raw cotton and activated carbon in the LRC. Importantly, FS concentrations remained unchanged in the LRC filtrate.

3.3.2. Immobilized yeast fermentation

When immobilizing cells onto a solid matrix such as calcium
alginate beads, a number of factors can affect the penetration of
cells into the bead and ultimately the conversion of FS to ethanol.
Factors affecting bead penetration include alginate content, the
ratio of yeast cells to alginate, and pore size. In a previous study,
these factors were optimized and we identified a suitable alginate
microlattice matrix for our bioethanol reactor, known as an eggbox structure [24].
Calcium alginate beads and cultured S. cerevisiae were used to
construct an ICR, with which we evaluated fermentation efficiency
using a number of different fruit waste hydrolysates. Total FS, ethanol concentrations, and FS-to-ethanol conversion rates were
obtained using two types of fermentation processes: ICR alone
and LRC followed by ICR (LRCICR). The volume metric ethanol
productivity (g/L/h) was calculated by dividing final ethanol concentration with respect to fermentation time (Table 5). The initial
FS concentrations in OP, MP, GP, LeP, LiP, MixP, and TFW were
57.5, 62.2, 46.7, 42.1, 31.0, 44.4, and 43.3 g/L, respectively. The relative FS concentrations decreased with increasing time in the LRC
ICR, especially over the first 9 h, whereas ethanol concentrations
increased. After 9 h, FS concentrations in OP, MixP, and TFW had
fallen to 2.4, 2.3, and 2.6 g/L, respectively, and ethanol concentrations had increased to 27.1, 29.5, and 20.3 g/L, respectively
(Fig. 4AC). When using ICR alone, without prior removal of D-limonene, FS concentrations were subsequently lower. After 9 h, FS
concentrations for OP, MixP, and TFW were 51.9, 17.9, and
20.8 g/L, respectively, whereas ethanol concentrations were 2.7,
15.9, and 12.9 g/L, respectively. In the LRCICR system, the FS-toethanol conversion rates for OP, MixP, and TFW feedstocks were
92.4%, 90.2% and 91.8%, respectively, after 10 d, whereas no further
ethanol production was observed in the ICR system after the first

I.S. Choi et al. / Applied Energy 140 (2015) 6574

9 h (Fig. 4D). These results are likely due to high D-limonene concentrations and its inhibitory effect on fermentation in the ICR system. Regarding the remaining feedstocks, MP and GP showed high
FS contents and low ethanol concentrations after ICR fermentation,
similar to the results obtained for OP, MixP, and TFW following ICR
fermentation. After ICR fermentation, LeP and LiP showed FS contents of 5.5 and 3.8 g/L, respectively, and ethanol concentrations
of 20.2 and 15.1 g/L, respectively (Supplementary Fig. S2AD).
The FS-to-ethanol conversion rates for LeP and LiP in the ICR system were only slightly lower compared to the LRCICR system
(Supplementary Fig. S2ad). This may have occurred because the
initial D-limonene concentrations in the LeP and LiP hydrolysates
were insufficient to inhibit fermentation. However, these results
indicate that the LRCICR fermentation system improved the FSto-ethanol conversion rates and ethanol concentrations even at
low D-limonene concentrations. Interestedly, the ethanol productivity of OP and MP, which are major citrus biomass sources, were
3.01 and 3.28 g/L/h, respectively, through the LRCICR system. In
other words, 1000 kg fresh OP and MP (19.8% and 20.1% of moisture contents) would be converted into 44.8 and 49.5 L of bioethanol, respectively (Table 6).
Several previous studies have examined the effects of pretreatment on CPW composition and subsequent bioethanol production;
however, ethanol concentrations and productivities obtained in
this study were similar to or greater than those observed in previous studies. For example, ethanol production from OP, using steam
explosion combined with acid pretreatment, produced 2527 g/L
ethanol concentration with around 0.5 g/L/h productivity [11].
Another study reported that the fermentation of MP and LeP after
steam explosion produced approximately 60 L/1000 kg (fresh matter) of ethanol concentration with 0.50.94 g/L/h productivity,
respectively [12,14].
Fruit waste and other solid residues, such as coffee waste and
rice, from agricultural by-products were considered bioethanol
production materials [13,21,33]. One main obstacle to achieving
efficient bioethanol production is the cost of production. The commercial success of ethanol production depends on productivity, in
terms of volume and concentration. Notably, our new process
achieved high ethanol production without costly pretreatment,
suggesting utility in industrial ethanol production applications.
The high ethanol production during the validation experiment
could be due to several factors, including suitable inhibitor
removal conditions, enzyme production, loading volume, and continuous yeast fermentation.
4. Conclusion
Fruit waste is an attractive biomass alternative for bioethanol
production because it has high levels of FS such as sucrose, glucose,
and fructose. In this study, these sugars were hydrolyzed and fermented without an energy-intensive conventional pretreatment.
After enzymatic hydrolysis with two in-house enzymes, D-limonene was removed using an adsorbent column containing raw cotton and activated carbon and directly conducted to an immobilized
reactor (LRCICR) for fermentation. Ethanol production in this
LRCICR system was 12-fold greater than that observed without
prior use of the sorbent column (LRC) to remove the fermentation
inhibiting D-limonene. This new approach to removing D-limonene
and enhancing immobilized yeast fermentation could potentially
be useful in more cost-effective bioethanol production.
Acknowledgements
This work was supported by Priority Research Centers Program
(2010-0020141) through the National Research Foundation of

73

Korea (NRF) funded by the Ministry of Education, Science and

Technology, and by a grant (S211314L010120) from Forest Science
& Technology Projects, Forest Service, Republic of Korea.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.apenergy.201
4.11.070.
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