Applied Energy 140 (2015) 65–74

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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
b
Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Mānoa, 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

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 a b s t r a c t

Article history: Large quantities of fruit waste are generated from agricultural processes worldwide. This waste is often
Received 22 March 2014 simply dumped into landfills or the ocean. Fruit waste has high levels of sugars, including sucrose, glu-
Received in revised form 17 November 2014 cose, and fructose, that can be fermented for bioethanol production. However, some fruit wastes, such as
Accepted 29 November 2014
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
Keywords:
peel, apple pomace, and pear waste) to produce bioethanol. Two in-house enzymes were produced from
Citrus peel waste
Bio ethanol
Avicel and CPW and were tested with fruit waste at 12–15% (w/v) solid loading. The rates of enzymatic
Enzymatic hydrolysis conversion of fruit waste to fermentable sugars were approximately 90% for all feedstocks after 48 h. We
D-Limonene extract also designed a D-limonene removal column (LRC) that successfully removed this inhibitor from the fruit
Continuous immobilized yeast fermentation waste. When the LRC was coupled with an immobilized cell reactor (ICR), yeast fermentation resulted in
ethanol concentrations (14.4–29.5 g/L) and yields (90.2–93.1%) that were 12-fold greater than products
from ICR fermentation alone.
Ó 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 petro-
leum) 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).
(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 compe-
tition 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 pro-
cessing. Citrus, which includes oranges, grapefruits, lemons, limes,

http://dx.doi.org/10.1016/j.apenergy.2014.11.070
0306-2619/Ó 2014 Elsevier Ltd. All rights reserved.
66 I.S. Choi et al. / Applied Energy 140 (2015) 65–74

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 25–40% 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, Euro-
pean 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 ferment-
able soluble sugars such as glucose, fructose, and sucrose along
with structural cellulose and hemicellulose. These chemical con-
stituents, along with the fact that fruit waste is in abundant supply,
suggest that fruit waste may be an excellent source of waste bio-
mass 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 byprod-
uct has high added value as a flavoring agent and for various
applications in the chemical industry. Thus, removing and recover-
ing 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
[7–14]. 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 poly-
mers). The most commonly used polymers are petrochemical poly-
mers 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.
 I.S. Choi et al. / Applied Energy 140 (2015) 65–74 67

Table 1
Citrus waste as substrate for bioethanol production.

Substrate Pretreatment Enzymesa Fermentation process Microorganism Ethanol production References

c
Orange peel Milling Pectinase, cellulase, glucosidase HF S. cerevisiae 4.7 [7]
Orange peel Milling Pectinase, cellulase, glucosidase HF Escherichia coli KO11 2.76c [8]
Orange peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 3.96c [9]
Orange peel Steam explosion Pectinase, cellulase, glucosidase SSF Kluyveromyces marxianus 3.45c [10]
Orange peel Acidic steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 2.7c [11]
Mandarin peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 59.3d [12]
Mandarin peel Popping Pectinase, xylanase, glucosidase SHEFb S. cerevisiae 46.2d [13]
Lemon peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 67.8d [14]
a
Commercial enzymes used for hydrolysis.
b
SHEF, separate hydrolysis and fermentation with vacuum evaporation.
c
Ethanol yields presented in%, w/v.
d
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]. Etha-
nol production from CPW has largely been conducted using com-
mercial 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 pec-
tinolytic enzymes [18]. Both fungal species are considered extra-
cellular 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 fer-
mentation 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 fer-
mentation. In addition, immobilizing the yeast cell wall confers
higher ethanol tolerance and cell concentrations, shorter fermenta-
tion 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 fer-
mentation 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 temper-
ature 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 inter-
nal standard. Gas chromatography (GC-2010, Shimadzu, Japan)
was used, and the analysis conditions, using a DB-225 capillary col-
umn (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 previ-
ous 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 col-
umn (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 Kor-
ean Culture Center of Microorganisms (KCCM), and Trichoderma
longibrachiatum (KCTC 6507) was purchased from the Korean Col-
lection for Type Cultures (KCTC). The lyophilized fungi were revi-
talized 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 Erlen-
meyer 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
68 I.S. Choi et al. / Applied Energy 140 (2015) 65–74

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 centri-
fuged and the supernatant was stored at 4 °C.
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 spe-
cific substrate solution consisting of 50 mM citrate phosphate buf-
fer, pH 4.8 (at 45 °C for 30 min), and appropriately diluted enzyme
concentrations. Endoglucanase (CMCase) and exoglucanase (Avice-
lase) 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 sug-
ars were quantified with dinitrosalicylic acid (DNS) at an absor-
bance 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 concentra-
tions of 12–16 and 10–25 mg protein/g fruit waste, respectively.
Enzymatic hydrolysis was performed on 1% matter (w/v) with cit-
rate phosphate buffer (pH 4.8) at 180 rpm for 48 h at 45 °C. Optimi-
zation of enzyme loading volume and the influence of biomass
hydrolysis time during enzymatic hydrolysis were measured using
HPLC as described in 2.2.
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 (100–300 mg) and activated-car-
bon (0–2 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 pro-
cess was conducted with- or without LRC on a fermentation reac-
tor. After fermentation, D-limonene was recovered from LRC using
hexane, and the recovery rate was determined using GC 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 con-
taining 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 algi-
nate 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 fer-
mentation of the CPW hydrolysate. ICR consists of a tubular col-
umn, constructed with a 2.1 cm ID and 25 cm length. About 70%
of the column was packed with immobilized yeast cells. The med-
ium 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 2.1 ± 0.0 5.6 ± 0.2 2.2 ± 0.0 2.4 ± 0.1 2.7 ± 0.1 5.6 ± 0.2 35.5 ± 0.5 12.1 ± 0.4 53.2 ± 0.4 68.2 ± 0.5
MP 2.9 ± 0.1 3.3 ± 0.1 2.4 ± 0.1 2.3 ± 0.1 3.9 ± 0.1 7.4 ± 0.2 39.4 ± 1.1 10.3 ± 0.8 57.1 ± 0.6 71.9 ± 0.9
GP 3.4 ± 0.0 4.8 ± 0.2 2.3 ± 0.1 2.2 ± 0.0 3.5 ± 0.2 1.4 ± 0.1 30.6 ± 0.8 8.2 ± 0.3 43.2 ± 0.7 59.4 ± 0.8
LeP 2.1 ± 0.1 5.2 ± 0.3 2.6 ± 0.2 2.1 ± 0.1 4.6 ± 0.1 ND 27.9 ± 0.4 3.3 ± 0.1 31.2 ± 0.4 47.8 ± 0.3
LiP 2.5 ± 0.2 8.5 ± 0.4 2.5 ± 0.1 2.0 ± 0.1 4.3 ± 0.1 ND 22.5 ± 1.2 0.7 ± 0.0 23.2 ± 1.0 43.0 ± 0.6
AP 1.7 ± 0.1 5.5 ± 0.1 6.2 ± 0.3 2.8 ± 0.1 4.2 ± 0.3 9.2 ± 0.1 25.2 ± 2.8 24.7 ± 0.3 59.1 ± 1.8 79.5 ± 1.5
BP 0.6 ± 0.1 4.4 ± 0.3 5.6 ± 0.4 3.6 ± 0.1 2.8 ± 0.1 ND 30.1 ± 0.8 15.2 ± 0.7 45.3 ± 0.3 71.5 ± 0.6
PP 1.3 ± 0.1 6.0 ± 0.3 20.2 ± 0.9 2.4 ± 0.0 4.5 ± 0.3 1.9 ± 0.1 21.1 ± 0.6 14.1 ± 0.5 37.1 ± 0.8 62.3 ± 0.7
MixP 2.7 ± 0.1 5.6 ± 0.2 2.4 ± 0.2 2.2 ± 0.0 3.8 ± 0.0 2.9 ± 0.1 32.0 ± 0.8 6.8 ± 0.3 41.7 ± 1.1 58.4 ± 0.8
TFW 2.0 ± 0.1 5.4 ± 0.4 5.5 ± 0.6 2.5 ± 0.1 3.8 ± 0.2 3.2 ± 0.3 29.0 ± 1.7 11.1 ± 0.9 43.4 ± 1.9 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.

Endoglucanase (U/mg protein) Exoglucanase (U/mg protein) Xylanase (U/mg protein) Pectinase (U/mg protein)
In-house enzyme A (HEA) 8.41 ± 0.11 0.18 ± 0.01 170.95 ± 1.81 17.90 ± 0.43
In-house enzyme B (HEB) 13.22 ± 1.21 1.26 ± 0.17 4.34 ± 0.52 1.11 ± 0.21
 I.S. Choi et al. / Applied Energy 140 (2015) 65–74 69

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

88.7 ± 1.6
88.4 ± 1.6
88.4 ± 2.1
70.9 ± 1.0
84.1 ± 2.0
packed-bed reactor column was 0.08 mL/min. The ICR was main-
tained in an incubator at 30 °C, and samples were withdrawn

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
aseptically from the bioreactor periodically during a 10-day per-
iod to analyze sugar and ethanol concentrations.

71.6 ± 2.2
85.7 ± 2.1
89.0 ± 1.2

89.4 ± 1.7
89.1 ± 0.9
Group B
3. Results and discussion

LeP
3.1. Fruit waste composition

79.6 ± 1.1
84.7 ± 1.9

91.2 ± 2.1
73.0 ± 1.0

91.1 ± 2.0
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.
The carbohydrate composition of the various fruit wastes dif-

TFW
fered as shown in Table 2. The total carbohydrate contents of the
fruit wastes were separated into soluble sugars, which dissolve

73.1 ± 1.7
84.1 ± 1.5
87.8 ± 2.2

90.4 ± 1.7
90.0 ± 2.0
easily in water, and insoluble sugars (cellulose and hemicellu-

MixP
lose) in the cell walls. Although arabinose and xylose were pres-
ent, they appeared in low concentrations in the fruit waste. We
mainly focused on fermentable sugars (FS), namely, glucose, fruc-

72.5 ± 1.6
80.1 ± 1.5
82.7 ± 1.7

90.3 ± 1.9
90.1 ± 1.0
tose, and sucrose. All fruit wastes presented were high in FS con-
tent (Table 2). Sucrose and fructose were present as soluble free

GP
sugars, whereas, glucose was part of the fruit waste structural
components and present as a free sugar. FS contents in the vari-

16 mg HEA /g fruit waste

73.1 ± 1.2
82.4 ± 2.9
86.1 ± 1.6
90.8 ± 1.2
90.7 ± 1.5
ous fruit wastes ranged from 23.2% to 59.1%. Orange peel (OP),
mandarin peel (MP), grapefruit peel (GP), apple pomace (AP),

MP
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

70.4 ± 2.2
83.4 ± 3.5
85.7 ± 2.1
90.2 ± 1.4
90.5 ± 2.0
Group A
31.2%, 23.2%, and 37.1%, respectively. FS contents in the CPW
mixture (MixP) and CPW, in combination with other fruit waste

OP
(TFW), were 41.7% and 43.4%, respectively.

70.6 ± 1.2

88.7 ± 1.2
88.9 ± 1.7
81.1 ± 0.8

88.9 ± 2.0
3.2. In-house enzyme production and fruit waste hydrolysis

LiP
The current cost of pretreatment and enzymes for biomass

70.1 ± 1.9

89.0 ± 2.1
89.1 ± 1.8
89.1 ± 2.1
82.1 ± 2.0
hydrolysis are major obstacles to large-scale ethanol production
Group B

[25]. The cost of cellulase is estimated at a minimum of $10/kg

LeP

protein [16]. Accordingly, it is necessary to reduce the cost and

amount of enzymes required for biomass hydrolysis to industrial-
ize the process. Here, we produced a suitable enzyme complex for
69.1 ± 1.7
72.8 ± 1.6
79.8 ± 2.1
84.1 ± 1.8
85.8 ± 3.5
fruit waste hydrolysis using CPW or Avicel as the carbon source.
TFW

In addition, we also report on the efficacy of the in-house enzyme

activities and fruit waste hydrolysis.
66.8 ± 1.5
75.4 ± 1.2
77.1 ± 3.3
76.2 ± 2.2
83.4 ± 1.1

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

MixP

hydrolysis
Between the two in-house enzymes evaluated (Table 3), HEA
exhibited the highest level of xylanase activity. Its pectinase
71.6 ± 1.5
73.6 ± 3.1
78.7 ± 1.6
80.1 ± 1.2
64.0 ± 0.9

activity was moderate, and both exoglucanase and endoglucan-

ase activities were observed. The activity of xylanase and pectin-
GP

ase were lower for HEB, compared to that of HEA, but

12 mg HEA/g fruit waste

endoglucanase and exoglucanase activities were higher or HEB.

65.8 ± 1.2

76.1 ± 1.8
81.2 ± 2.2
82.4 ± 3.3
73.1 ± 0.9

Interestingly, HEA was produced using CPW as the carbon source,

and it showed high xylanase activity. This may have occurred
MP

because hemicellulose forms a large component of the polysac-

charides in CPW [13,26], and xylanase produced monosaccha-
63.2 ± 1.8
72.1 ± 1.2
75.4 ± 3.1
80.4 ± 2.2
81.2 ± 2.6
Group A

rides by CPW hydrolysis for fungal survival. Microorganisms

produce the appropriate complex enzymes for hydrolysis during
OP

growth on a given substrate. The presence of hemicellulose-

derived saccharides in CPW is thought to be important for HEA
Mg HEB/g fruit waste

induction. Based on the above mentioned HEA and HEB activities,

Conversion rate (%)

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,
Table 4

15

25
10

20
0

GP, LeP, LiP, MixP, and TFW substrates. Data for the hydrolysis
of various fruit wastes by HEA and HEB are shown in Table 4.
70 I.S. Choi et al. / Applied Energy 140 (2015) 65–74

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) 3 6 9 12 15 18 21 24 48
Group A (12%) OP 53.0 ± 2.3 64.0 ± 2.0 76.2 ± 2.7 84.8 ± 3.1 86.2 ± 1.3 87.3 ± 1.8 88.8 ± 2.0 89.5 ± 2.2 90.2 ± 1.7
MP 51.8 ± 1.9 66.3 ± 3.0 78.8 ± 2.1 85.7 ± 2.7 87.4 ± 3.1 88.5 ± 2.0 89.3 ± 3.3 89.7 ± 2.6 90.8 ± 2.1
GP 51.8 ± 2.5 65.3 ± 3.1 75.6 ± 2.9 85.7 ± 2.1 87.4 ± 3.5 88.5 ± 2.5 89.3 ± 2.5 89.7 ± 3.0 90.1 ± 2.3
MixP 51.8 ± 1.5 65.0 ± 2.4 76.3 ± 2.3 85.7 ± 2.4 87.4 ± 2.7 88.5 ± 3.0 89.3 ± 3.2 89.7 ± 2.8 90.0 ± 1.8
TFW 52.1 ± 2.1 67.2 ± 2.0 79.1 ± 2.6 87.8 ± 2.1 89.2 ± 2.3 90.1 ± 1.8 90.3 ± 2.7 90.8 ± 2.1 91.4 ± 2.1
Group B (15%) LeP 45.8 ± 2.1 61.2 ± 2.6 72.4 ± 1.9 80.1 ± 2.6 85.3 ± 1.9 87.0 ± 2.2 87.8 ± 1.7 89.3 ± 2.6 89.0 ± 1.7
LiP 47.2 ± 2.9 64.3 ± 1.9 73.8 ± 1.9 79.4 ± 3.0 86.7 ± 2.7 87.8 ± 1.9 88.0 ± 1.9 88.0 ± 2.4 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 LRC–ICR system.

Fermentable sugar content (g/L) Enzymatic hydrolysate (g/L, %a) Ethanol concentration (g/L) Ethanol yield (%) Productivity (g/L/h)
OP 63.8 57.5/90.2% 27.1 92.4 3.01
MP 68.5 62.2/90.8% 29.5 93.1 3.28
GP 51.8 46.7/90.1% 21.6 90.7 2.40
MixP 50.0 44.4/90.0% 20.4 90.2 2.27
TFW 47.4 43.3/91.4% 20.3 91.8 2.26
LeP 46.8 42.1/89.0% 19.6 91.1 2.18
LiP 34.8 31.0/88.7% 14.4 90.8 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 species-
dependent, the relatively low loading of HEA supplemented with
HEB achieved a high overall hydrolysis rate. An increase in FS con-
centrations 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 differ-
ent 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 hemicel-
lulose 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 pro-
duction. This is because S. cerevisiae shows preferential consump-
tion 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) 65–74 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 effi-
ciency and avoid limitations, sucrose must be hydrolyzed to glu-
cose 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 var-
ied 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 sub-
strate 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 fermenta-
tion. 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 inhib-
its 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 feed-
stock may decrease to as low as 10% after pretreatment due to
72 I.S. Choi et al. / Applied Energy 140 (2015) 65–74

Fig. 4. Comparisons of fermentable sugar conversion and ethanol concentrations in ICR vs. LRC–ICR 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 LRC–ICR ( ) fermentation, and gray solid lines indicate the amount of ethanol produced from ICR ( ) or LRC–ICR ( ) fermentation.

losses resulting from the Maillard reaction, caramelization, and
oxidation [13,31–33]. With the aim of developing a more cost-
effective, 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 concen-
trations 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. How-
ever, 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 0.321–1.858% (w/w) D-limonene (Fig. 3B);
however, after passing through the LRC, D-limonene was undetect-
able. 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 con-
centrations, between 0.05% and 0.15% (v/w), that can affect the fer-
mentation 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 sur-
face of sorbents is inversely proportional to oil viscosity and con-
centration [34], adsorption should be high in the pores of the
raw cotton and activated carbon in the LRC. Importantly, FS con-
centrations 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 egg-
box 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, eth-
anol concentrations, and FS-to-ethanol conversion rates were
obtained using two types of fermentation processes: ICR alone
and LRC followed by ICR (LRC–ICR). The volume metric ethanol
productivity (g/L/h) was calculated by dividing final ethanol con-
centration 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 rel-
ative 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 concentra-
tions had increased to 27.1, 29.5, and 20.3 g/L, respectively
(Fig. 4A–C). When using ICR alone, without prior removal of D-lim-
onene, 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 LRC–ICR system, the FS-to-
ethanol 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) 65–74
9 h (Fig. 4D). These results are likely due to high D-limonene con-
centrations and its inhibitory effect on fermentation in the ICR sys-
tem. 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 con-
tents of 5.5 and 3.8 g/L, respectively, and ethanol concentrations
of 20.2 and 15.1 g/L, respectively (Supplementary Fig. S2A–D).
The FS-to-ethanol conversion rates for LeP and LiP in the ICR sys-
tem were only slightly lower compared to the LRC–ICR system
(Supplementary Fig. S2a–d). 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 LRC–ICR fermentation system improved the FS-
to-ethanol conversion rates and ethanol concentrations even at
low D-limonene concentrations. Interestedly, the ethanol produc-
tivity of OP and MP, which are major citrus biomass sources, were
3.01 and 3.28 g/L/h, respectively, through the LRC–ICR system. In
other words, 1000 kg fresh OP and MP (19.8% and 20.1% of mois-
ture contents) would be converted into 44.8 and 49.5 L of bioetha-
nol, respectively (Table 6).
Several previous studies have examined the effects of pretreat-
ment 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 previ-
ous studies. For example, ethanol production from OP, using steam
explosion combined with acid pretreatment, produced 25–27 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 mat-
ter) of ethanol concentration with 0.5–0.94 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 com-
mercial 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 con-
tinuous 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 fer-
mented without an energy-intensive conventional pretreatment.
After enzymatic hydrolysis with two in-house enzymes, D-limo-
nene was removed using an adsorbent column containing raw cot-
ton and activated carbon and directly conducted to an immobilized
reactor (LRC–ICR) for fermentation. Ethanol production in this
LRC–ICR 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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