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Applied Energy
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Department of Wood Science and Landscape Architecture, Chonnam National University, Gwangju 500-757, Republic of Korea
Faculty of Biotechnology, College of Applied Life Sciences, Cheju National University, Jeju 690-756, Republic of Korea
c
Bio-Energy Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea
d
Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea
b
a r t i c l e
i n f o
Article history:
Received 23 December 2011
Received in revised form 28 March 2012
Accepted 31 March 2012
Available online 26 April 2012
Keywords:
Popping pretreatment
Ethanol productivity
Mandarin peel waste
Limonene
a b s t r a c t
In this study, we designed a biomass popping pretreatment, system using a red burner and a horizontal
cylinder rotating on an axis, to produce ethanol from mandarin (Citrus unshiu) peel (MP) waste. Popping
pretreatment was performed at 150 C for 10 min without chemical treatment. Popping pretreatment
reduced the size of particles to less than 1 mm and decreased the concentration of D-limonene, a yeast
fermentation inhibitor, from 0.21% to 0.01%. Enzymatic hydrolysis of pretreated MP was performed in
50 mM sodium acetate buffer (pH 4.8) at 45 C for 6 h, and the total saccharication rate was approximately 95.6%. The vacuum evaporation process increased the fermentable sugar concentration to 10%
(glucose 7.1% and fructose 2.9%). Subsequent fermentation at 30 C at pH 5.0 for 12 h in a laboratory bioreactor increased the ethanol yield to 90.6%, compared to 78% at 36 h from raw MP.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Bioethanol is a promising alternative to fossil fuels because it is
a renewable resource. It is a liquid fuel that can be adapted to
existing fuel supply systems and can replace fossil fuels in the
transportation sector [14]. However, starch-based bioethanol production is criticized because starch is generally derived from food.
Thus, the use of starch as a source of bioethanol can reduce food
supplies and increase food price. The use of starch- or sugar-rich
feedstock has directly increased food and bioethanol prices due
to the high price of raw materials, which account for 4075% of
the total expenses for ethanol production [5]. Therefore, alternative biomass sources are required for bioethanol production, such
as agricultural byproducts, forest residues, or energy crops (lignocellulosic biomass) [614].
Citrus peel waste is a suitable candidate for bioethanol production because it contains low lignin and high soluble sugar concentrations [15,16]. Citrus peel waste is rich in fermentable sugars
such as glucose, fructose and sucrose, along with insoluble polysaccharides such as cellulose and hemicellulose [16]. According
to the USDA [17], world citrus production was estimated at 49.9
million tons during the 2009/2010 growing season. After juice
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).
0306-2619/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.apenergy.2012.03.066
205
then freeze-dried. The samples were then coated with gold (20 nm)
in a sputter coater prior to being observed and photographed with
a scanning electron microscope (S2400, Hitachi, Japan).
2.4. D-limonene extraction
Extraction and quantication of D-limonene were performed
according to the method described previously [25]. Matters (raw
and popping-pretreated) were homogenized in 10 mL of hexane
and known amount of camphor as an internal standard. After treatment for 3 h, the residue was discarded and 5 mL of supernatant
was transferred to a tube. 0.2 mL of 2 N KOH in methanol was
added and the samples were mixed for 1 min. After the addition
of 1 mL of distilled water, the samples were vigorously mixed
and then centrifuged for 5 min at 3000 rpm. The hexane phase
was analyzed by gas chromatography (CP-9100, Chrompack) using
a CP-Sil 5 CB fused silica capillary column (25 m 0.32 mm i.d.,
1.2 lm lm thickness, Chrompack) operated with He, injector temperature 280 C, ame ionization detector (FID) at 280 C.
2.5. Enzymatic hydrolysis
Enzymatic hydrolysis was performed on matter with individual or mixed commercial cellulase (Celluclast 1.5L), pectinase
(Pectinex SP-L), xylanase (X2629 endo-1,4-b-D-xylanase), and bglucosidase (Novozym 188) at 45 C for 24 h as follow previous
studies [8,19]. Cellulase, pectinase and b-glucosidase were purchased from Novozyme A/S (Bagsvaerd, Denmark) and xylanase
was provided by Sigma (St. Louis, MO, USA). Cellulase and pectinase activities were determined as shown previously [19] and
xylanase activity was measured according to Bae et al. [27]. The
activities of cellulase, pectinase and xylanase were 0.122 lter paper unit (FPU)/mg protein, 240 international units (IU)/mg protein
and 2.65U/mg xylanase, respectively. The activity of b-glucosidase
was 2.6 IU/mg solid as reported by the supplier.
The raw and popping pretreated matters were diluted in 15 mL
test tubes to 1% (w/v) dry matter with 50 mM sodium acetate buffer (pH 4.8). The optimal enzyme cocktails for measuring the sugar
composition of MP was determined with different enzyme cocktails on raw or pretreated MP as compare with non enzyme treated
(control). Based on a small scale study, the enzymatic hydrolysis
system was scaled up to a 5 L bioreactor (1% dry matter).
2.6. Sugars analysis
After complete hydrolysis, all samples in test tube with cap
were boiled at 105 C for 5 min to inactivate enzymes and then
centrifuged for 20 min at 13,000 rpm and 26,000g (Avanti JE,
Beckman, USA) before analyzing the soluble sugars. The sample
supernatants were analyzed for soluble sugar content using high
performance liquid chromatography (HPLC) with a refractive index
detector (2414, Waters, USA). The mixtures were treated with
50 ll of enzyme and loaded into the REZEX RPM (Phenomenex,
USA) column (300 7.8 mm) at 85 C and eluted with deionized
water at a ow rate of 0.6 mL per min.
The insoluble solids were washed ve times in distilled water
and pelleted. Pellets were lyophilized before analyzing the sugars.
Each pelleted samples were treated using a modied alditol acetate method and the neutral sugar contents were measured by
gas chromatography (GC) [28]. Gas chromatography analysis conditions were as follows, gas chromatography (GC-2010, Shimadzu,
Japan) using a DB-225 capillary column (30 m 0.25 mm i.d.,
0.25 lm lm thickness, J&W) operated with He, injector temperature of 220 C, ame ionization detector (FID) at 250 C, and
oven temperature programming: 100 C for 1.5 min, 5 C/min to
220 C.
206
Popping pretreatment uses whole MP without chemical treatments or mechanical cuts. This procedure simplies the MP pretreatment compared to other methods such as steam explosion.
Generally, steam explosion requires diluted chemicals (sulfuric
acid or sodium carbonate) or sample cutting to produce ethanol
from citrus species [15,18].
We evaluated the observed structural and chemical changes
through the popping pretreatment on MP. The D-limonene concentration was also analyzed before and after popping pretreatment
because terpenoid is a fermentation inhibitor. To enhance ethanol
production, MP required a pretreatment process that effectively
disrupts cell walls, increasing access of polysaccharides to the enzymes, as reported previously [29].
SEM images indicated that popping treatment reduced MP
particle size, and consequently increased the substrate surface
area (Supplementary Fig. S3A and B). The increased cell wall surface area improved accessibility of cell wall-degrading enzymes,
and the reduced particle size was economically benecial.
Mechanical pretreatments are generally applied during bioethanol production. A particle size reduction to 251 lm improves
the hydrolysis yield by 525% [30,31]. However, due to the high
energy requirement, mechanical pretreatments are not economically feasible [32]. According to Carroll and Somerville [7], a
hardwood particle size of 1.6 mm required 130 kWh/kg, whereas
grass straws required 7.5 kWh/kg. In comparison, an approximately 10 10 cm MP particle was reduced to 1 mm after popping pretreatment.
The MP cell wall carbohydrate concentration was affected by
popping pretreatment (Table 1). GC analysis of the MP cell wall
carbohydrate showed glucose and xylose as the primary components with concentrations of 24.8% and 3.1%, respectively. There
Table 1
Chemical composition of raw, popping-pretreated MP and various lignocellulosic feedstocks. Organic solvent extractives, lignin, and ash were analyzed by TAPPI standard method
and monosaccharide was analyzed by using GC. The values for raw and popping pretreated mandarin peel from analysis of this study.
(% of dry matter)
Extractives
Rhamnose
Arabinose
Xylose
Mannose
Galactose
Glucose
Lignin
Ash
38.3
44.3
nd
3.8
12.9
17.0
3.8
0.5
0.9
nd
nd
nd
11.2
1.7
8.4
2.1
2.4
2.8
3.1
5.8
3.7
22.1
19.2
20.4
2.0
1.7
3.0
0.4
0.3
0.3
4.2
1.9
6.4
0.5
0.8
0.9
24.8
32.0
25.8
39.0
32.6
31.0
8.6
9.2
nd
23.1
16.9
17.6
4.2
3.1
nd
3.7
10.2
5.8
nd = no data.
a
From Hendriks and Zeeman [32] for citrus peels from French citrus industrial factory.
b
From Carroll and Somerville [7].
Table 2
Concentration of saccharide as soluble sugars by enzymatic hydrolysis on raw and popping pretreatment material. Each enzymes loading follow as; pectinase 5 mg/g dry matter,
xylanase 5 mg/g dry matter and b-glucosidase 2 mg/g dry matter. Abbreviations used monosaccharides (Suc; sucrose, Glu; glucose, Gal; galactose, Ara; arabinose, Fruc; fructose)
and enzymes (Pec; pectinase, Xyn; xylanase, b-G; b-glucosidase.).
Content (g/10 g)
Enzyme cocktails
Suc
Glu
Gal
Ara
Fruc
Total
Raw
Cont.
Pec
Xyn
Pec + Xyn
Pec + Xyn + b-G
0.94
0.00
0.46
0.00
0.00
1.73
3.10
3.99
4.71
4.60
0.04
0.11
0.03
0.12
0.00
0.00
0.07
0.07
0.10
0.09
1.52
1.90
1.77
2.21
2.16
4.23
5.18
6.31
7.14
6.84
Popping pretreatment
Cont.
Pec
Xyn
Pec + Xyn
Pec + Xyn + b-G
0.04
0.00
0.09
0.00
0.00
1.75
3.17
4.05
4.42
4.51
0.02
0.18
0.03
0.15
0.12
0.10
0.24
0.19
0.26
0.28
1.47
1.45
1.55
1.33
1.84
3.39
5.04
5.91
6.15
6.76
207
Table 3
Carbohydrate concentration of solid residues from MP (raw and popping pretreatment) after enzymatic hydrolysis by GC. Abbreviations used, monosaccharides (Rham;
rhamnose, Ara; arabinose, Xyl; xylose, Man; mannose, Gal; galactose, Glu; glucose) and enzymes (Pec; pectinase, Xyn; xylanase, b-G; b-glucosidase.).
Content (g/kg)
Enzyme cocktails
Rham
Ara
Xyl
Man
Gal
Glu
Total
Raw
Cont.
Pec
Xyn
Pec + Xyn
Pec + Xyn + b-G
3.8
0.5
3.0
0.3
0.8
11.2
0.5
1.1
0.1
0.2
3.1
1.0
0.9
0.7
0.8
2.0
0.3
0.2
0.1
0.4
4.2
0.3
0.3
0.1
0.2
24.8
4.3
4.4
1.2
4.3
49.1
6.9
9.9
2.5
6.7
Popping pretreatment
Cont.
Pec
Xyn
Pec + Xyn
Pec + Xyn + b-G
0.5
0.1
0.1
0.1
0.0
1.7
0.3
0.4
0.2
0.2
5.8
0.8
0.6
0.6
0.8
1.7
0.2
0.2
0.1
0.1
1.9
0.2
0.1
0.1
0.0
32.0
3.2
1.5
1.3
0.8
43.6
4.8
2.9
2.4
1.9
Fig. 1. Time course changes in glucose conversion rate (A) and carbohydrate concentration (B) during enzymatic hydrolysis.
208
Fig. 2. Limonene concentration of MP and orange peel (raw and popping pretreated). MP Raw; raw mandarin peel, MP Popping; popping-pretreated mandarin
peel, OP Raw; raw orange peel, OP Popping; popping-pretreated orange peel.
Popping pretreatment applied to orange peel decreased D-limonene concentration
from 1.70% to 0.02%.
may be related to disruption of yeast cellular membranes and subsequent H+ and K+ transport in glycolysis. D-limonene concentration decreased after popping pretreatment. Raw MP contains
0.21% D-limonene, but its concentration was reduced to 0.01% after
popping pretreatment (Fig. 2). Interestingly, the D-limonene
concentration decreased in orange peel (from 1.70% to 0.02%), possibly below the fermentable concentration [18,19].
Separate hydrolysis and fermentation (SHF), semi-simultaneous
saccharication and fermentation (SSSF), and simultaneous saccharication and fermentation (SSF) are generally considered to
be the primary methods for bioethanol production [39]. We modied SHF by including vacuum evaporation (SHEF).
A 10% (glucose 7.1%, fructose 2.9%) sugar solution was obtained
using vacuum evaporation from low concentrate hydrolysates.
After vacuum evaporation, fermentation was performed in a fermentor containing 1 L of fermentation medium inoculated with
10% high density cell inoculum. Ethanol yields were based on the
total fermentable sugars of both raw and pretreated MP. For raw
MP, fermentation peaked at 36 h when ethanol concentration
and yield efciency were 39.8 g/L and 78%, respectively. The ethanol yield of raw MP was lower than pretreated MP because the
concentration of D-limonene was higher than after pretreatment.
In contrast to raw MP, the fermentation of pretreated MP hydrolysate was almost complete after 12 h with 46.2 g/L ethanol concentration and 90.6% ethanol yield. More interestingly, for ethanol
productivity, pretreated MP fermentation produced 3.85 g/L h,
while raw MP fermentation produced 1.11 g/L h.
According to a previous study on the fermentation of orange
peel or MP wastes, 2448 h fermentation is required to achieve
over 90% ethanol yield [15,19,34,35,40]. Our process achieved a
greater ethanol yield with a much shorter fermentation time,
which is important for industrial applications because it reduces
the overall production cost. The high ethanol productivity and
yield efciency in our study could be due to several factors, including suitable hydrolysate concentrations, optimized pH, temperature, and low concentrations of D-limonene.
3.4. Overall mass balance
We used an overall mass balance diagram to describe the popping-pretreated MP and conversion of carbohydrates to ethanol
by enzymatic hydrolysis (Fig. 3). Compared to the raw MP values,
popping pretreatment increased the solid glucose concentration
by 22.5%, while D-limonene concentration decreased from 0.21%
to 0.01%. During enzymatic hydrolysis of pretreated MP, 311.7 g
of glucose were hydrolyzed from an initial 320 g of solid glucose,
while 8.3 g of residual glucose remained as unhydrolyzed cellulose.
The mass balance for glucose after enzymatic hydrolysis was 97.5%.
Hydrolysate was concentrated to 10%, and 93.6 L of water was
recovered by vacuum evaporation. Fermentation using S. cerevisiae
effectively converted monomeric glucose and fructose with 90.6%
fermentation yield and 373 mL of ethanol. These results suggest
that popping pretreatment combined with vacuum evaporation is
an acceptable industrial process for ethanol production from MP.
4. Conclusion
Mandarin (C. unshiu) peel waste is an attractive source of biomass for bioethanol production and has several advantages over
lignocellulosic biomass rich in hemicellulose and lignin (Table.
1). Popping pretreatment ruptures MP cell walls, producing particles with diameters less than 1 mm and also reduces D-limonene
concentrations to less than 0.01%. The pretreated MP does not yield
higher sugar concentrations in the hydrolysate after enzymatic
treatment but signicantly reduces D-limonene content. By com-
209
210
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