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Bioresource Technology 101 (2010) 42464250

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Production of biofuels, limonene and pectin from citrus wastes


Mohammad Pourbafrani a,b,*, Gergely Forgcs a,b, Ilona Srvri Horvth b, Claes Niklasson a,
Mohammad J. Taherzadeh b
a
b

Chemical Reaction Engineering, Chalmers University of Technology, 412 96 Gteborg, Sweden


School of Engineering, University of Bors, 501 90 Bors, Sweden

a r t i c l e

i n f o

Article history:
Received 16 November 2009
Received in revised form 14 January 2010
Accepted 20 January 2010
Available online 9 February 2010
Keywords:
Citrus waste
Ethanol
Biogas
Limonene
Pectin

a b s t r a c t
Production of ethanol, biogas, pectin and limonene from citrus wastes (CWs) by an integrated process
was investigated. CWs were hydrolyzed by dilute-acid process in a pilot plant reactor equipped with
an explosive drainage. Hydrolysis variables including temperature and residence time were optimized
by applying a central composite rotatable experimental design (CCRD). The best sugar yield (0.41 g/g
of the total dry CWs) was obtained by dilute-acid hydrolysis at 150 C and 6 min residence time. At this
condition, high solubilization of pectin present in the CWs was obtained, and 77.6% of total pectin content
of CWs could be recovered by solvent recovery. Degree of esterication and ash content of produced pectin were 63.7% and 4.23%, respectively. In addition, the limonene of the CWs was effectively removed
through ashing of the hydrolyzates into an expansion tank. The sugars present in the hydrolyzates were
converted to ethanol using bakers yeast, while an ethanol yield of 0.43 g/g of the fermentable sugars was
obtained. Then, the stillage and the remaining solid materials of the hydrolyzed CWs were anaerobically
digested to obtain biogas. In summary, one ton of CWs with 20% dry weight resulted in 39.64 l ethanol,
45 m3 methane, 8.9 l limonene, and 38.8 kg pectin.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
World production of citrus fruits is over 88 million tons per year
(Marin et al., 2007). Almost half of these fruits is squeezed to juice,
and the remainder including peel, segment membranes and other
by-products is considered as citrus wastes (CWs) (Wilkins et al.,
2007a). These CWs can be dried and used as raw material for pectin
extraction or pelletized for animal feed (Mamma et al., 2008).
However, a large fraction of CWs is still deposited every year. This
deposition is not favored due to both economic and environmental
arguments such as high transportation costs, lack of disposal sites,
and the land-lling material having high organic content (Tripodo
et al., 2004).
CWs contain different carbohydrate polymers, which makes
them interesting sources for production of biogas and ethanol
(Gunaseelan, 2004; Mizuki et al., 1990; Pourbafrani et al., 2007;
Wilkins et al., 2007b). The main obstacle to using CWs as a substrate for biogas production is the presence of limonene in CWs.
This component is very toxic for digesting microorganisms and decreases the biogas yield (Mizuki et al., 1990). Limonene is also a

* Corresponding author. Address: Chemical Reaction Engineering, Chalmers


University of Technology, 412 96 Gteborg, Sweden. Tel.: +46 33 435 43 61; fax:
+46 33 436 4008.
E-mail address: pour@chalmers.se (M. Pourbafrani).
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.01.077

strong inhibitor for microorganisms in ethanol production


(Pourbafrani et al., 2007; Wilkins et al., 2007b). Therefore, this
component should be separated from the CWs prior to digestion
or fermentation steps. It should also be noticed that digesting bacteria can hydrolyze the carbohydrate polymers in CWs and convert
them nally to biogas, while hydrolysis by enzymes or chemicals is
necessary to convert these polymers to sugars and then ferment
the sugars by e.g. bakers yeast to ethanol.
Two alternative processes for production of ethanol from CWs
based on enzymatic hydrolysis have been previously introduced
(Stewart et al., 2005; Wilkins et al., 2007b). In the rst alternative
(Stewart et al., 2005), CWs were hydrolyzed using a mixture of enzymes (cellulose, pectinase and b-glucosidase). Then, limonene
was removed from hydrolyzate by ltration and ethanol was produced from fermentable sugars. In the second alternative (Wilkins
et al., 2007b), limonene was partly released using steam stripping.
Then, carbohydrate polymers were hydrolyzed and fermented to
ethanol through a simultaneous saccharication and fermentation
(SSF) process. In both alternatives, non-fermentable sugars and the
residue of solid polymers were dried to be used as cattle feed
(Stewart et al., 2005). Application of these alternatives is hampered
by the high cost of enzyme and the slow rate of hydrolysis reactions (Grohmann et al., 1995). In addition, mechanical pretreatment of biomass, and high demand for energy in distillation and
drying processes, might considerably increase the cost of the
process.

M. Pourbafrani et al. / Bioresource Technology 101 (2010) 42464250

A process based on dilute-acid hydrolysis of CWs can be considered as another alternative (Grohmann et al., 1995; Talebnia et al.,
2008). However, the data are limited to the lab-scale experiments
and it is difcult to scale up the process to industrial scale. In both
studies (Grohmann et al., 1995; Talebnia et al., 2008), mechanical
pretreatment was used before hydrolysis and the experiments
were carried out in low solid/liquid ratio (Grohmann et al.,
1995). Furthermore, dilute-acid hydrolysis showed low yields of
sugars from the carbohydrate polymers.
The aim of the current work was to introduce a new process for
production of ethanol and biogas from CWs. The CW was pretreated with dilute-acid explosion process to hydrolyze the CWs
and also to get rid of limonene. The resultant slurry was then centrifuged, and the liquid part was fermented to ethanol and distilled.
The stillage from the distillation column and the remained solids
were mixed and digested to biogas. An industrial conguration
was suggested for this process.
2. Methods
2.1. Citrus wastes composition
The CWs used in this work was the residue of orange obtained
from Brmhults juice factory (Bors, Sweden) and stored frozen at
20 C until use. Total dry content of CW was determined by drying at 110 C for 48 h and it was 20.00 0.80% w/w. The composition of the CWs used in this work as percentage of dry matter was:
glucose 8.10 0.46; fructose 12.00 0.21; sucrose 2.80 0.15; pectin 25.00 1.20; protein 6.07 0.10; cellulose 22.00 1.95; hemicellulose 11.09 0.21; ash 3.73 0.20; lignin 2.19 0.04 and
limonene 3.78 0.30.
2.2. Dilute-acid hydrolysis
A 10-L high-pressure reactor (Process & Industriteknik AB, Sweden) was used for dilute-acid hydrolysis. The CWs was diluted with
distilled water to obtain 2 kg slurry with 15% total solid content.
Sulfuric acid (98%) was added to the slurries to reach nal concentration of 0.5% v/v. The slurries were then hydrolyzed at various
temperatures of 130, 150 or 170 C with different residence times
of 3, 6 and 9 min according to the experimental design (Table 1). A
central composite rotatable design (CCRD) was used to design the
experimental setup and to optimize hydrolysis variables including
temperature and time (Talebnia et al., 2008). The reactor was
heated with direct injection of 60-bar steam, provided by a power

Table 1
Experimental design of hydrolysis temperature and time in dilute-acid hydrolysis of
CWs and the total sugars yield of each hydrolysis (actual and predicted values based
on the model in Eq. (1)).
Test no.

1
2
3
4
5
6
7
8
9
10
11
12
13
a

Variables

YTSa

YTSa

T (C)

Time (min)

Actual

Predicted

130
170
130
170
130
170
150
150
150
150
150
150
150

3
3
9
9
6
6
3
9
6
6
6
6
6

26.49
33.73
30.41
33.00
28.60
36.85
35.65
37.55
42.05
40.54
41.09
41.58
41.40

25.38
33.73
29.40
33.10
30.71
36.74
36.75
38.45
40.93
40.93
40.93
40.93
40.93

YTS = yield of total sugars.

4247

plant located in Bors, Sweden. The hydrolyzed slurry was then


explosively discharged to an atmospheric pressure expansion tank
to cool down. The materials were then centrifuged at 11,500g for
10 min to separate solid from the hydrolyzate supernatant. The solid residue was washed four times with 200 ml distilled water to
separate the possible remaining sugars. The washing water was
added to the hydrolyzate supernatant, which was neutralized
and fermented. All experiments were duplicated and the standard
deviations were less than 3%.
2.3. Yeast strain and fermentation
The yeast Saccharomyces cerevisiae ATCC 96581, obtained from
LGC standards (Sweden), was used in the fermentation experiment.
The strain was maintained on agar plates made from yeast extract
(10 g/l), soy peptone (20 g/l), and agar (20 g/l) with D-glucose
(20 g/l) as an additional carbon source. The inoculum culture was
grown in 250-ml cotton-plugged conical asks on a shaker at
30 C for 24 h. The liquid volume was 100 ml and the growth medium was a dened synthetic medium (Taherzadeh et al., 1996)
including 50 g/l glucose as carbon and energy source. Anaerobic
fermentation was carried out by adding 50 ml of inoculum culture
to the hydrolyzate media in a bioreactor (Biostat A., B. Braun Biotech, Germany). The liquid volume of the reactor was 1 l. Temperature, stirring rate and pH were controlled at 30 C, 200 rpm and 5,
respectively. Nitrogen gas was steadily sparged at the rate of
600 ml/min in order to assure anaerobic conditions inside the
reactor.
2.4. Pectin recovery and analysis
The hydrolyzate supernatant (after the centrifugation) was ltered two times by lter paper to completely remove insoluble
materials. The pH was then increased from 1.2 to 2.2 and an equal
volume of 96% ethanol was added to precipitate pectin from the
solution at room temperature within 4 h. The precipitate was separated by centrifugation at 180g for 60 min and washed ve times
with ethanol (45%) according to a previous procedure (Faravash
and Ashtiani, 2007), and then dried at 50 C. The degree of esterication (DE) of the pectin was determined by the Fourier transform infrared (FTIR, Nicolet Instrument Corporation, USA) as
described before (Faravash and Ashtiani, 2007). A spectral resolution of 4 cm1 with 100 scan was applied to obtain the peak position and peak area with precise accuracy. The ash content of pectin
was measured by heating the pectin at 660 C for 8 h (Faravash and
Ashtiani, 2007).
2.5. Digestion
The stillage was obtained by heating the fermented hydrolyzate
at 96 C in order to evaporate ethanol. The stillage was then mixed
with the solid residue out of the hydrolysis reactor, which was already centrifuged and washed. The mixture was then neutralized
and used as substrate for digestion. Volatile solid (VS) of this substrate was measured by the loss on ignition of the dried sample at
550 C. It was adjusted to 3 g VS/100 g substrate by adding distilled
water. The active inoculum was supplied from a municipal waste
digester (Bors, Sweden) operating at 55 C.
Two-liter glass bottles with a thick rubber septum were used as
reactors (Hansen et al., 2004). Each reactor was fed with 200 g substrate (3% VS) and 400 g inoculum (about 1% VS), and ushed with
a gas containing 80% N2 and 20% CO2 to ensure anaerobic conditions (Hansen et al., 2004). The reactors were incubated at 55 C
for 50 days, while shaking twice a day. Three blanks with only
water and inoculum were used to measure the methane produc-

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M. Pourbafrani et al. / Bioresource Technology 101 (2010) 42464250

tion originating from the inoculum. All the digestion experiments


were carried out in triplicate.
2.6. Analytical methods
An Aminex HPX-87P ion-exchange column (Bio-Rad, USA) was
used at 85 C for measuring sucrose, glucose, galactose, arabinose
and fructose concentrations. Ultra-pure water was used as eluent
at a ow rate of 0.4 mL/min. Ethanol, succinic acid and glycerol
concentrations were determined on an Aminex HPX-87H column
(Bio-Rad, USA) at 60 C using 5 mM H2SO4 at a ow rate of
0.6 ml/min. A refractive index (RI) detector (Waters 2414, Milipore,
Milford, USA) and UV absorbance detector at 210 nm (Waters
2487) were used in series. Succinic acid was analyzed from UV
chromatograms while the residue of metabolites was quantied
from the RI chromatograms.
Releasing the limonene from CW was carried out using a mixture of cellulase, pectinase and b-glucosidase enzymes (Pourbafrani et al., 2007; Talebnia et al., 2008). The concentration of limonene
was determined by addition of n-heptane (99% purity) to the
hydrolyzate with a ratio of 1/5 and centrifugation at 3500g for
30 min to extract the oil. The resulting supernatant was then analyzed by a GCMS (HewlettPackard G1800C, Agilent, Palo Alto,
CA) where the carrier gas was helium. The temperature was initially 50 C and was increased to 250 C at the rate of 15 C/min
and maintained at this temperature for 3 min (Pourbafrani et al.,
2007).
Gas samples from the digesting reactors were taken by a
0.25 ml glass syringe (VICI, Precision Sampling Inc., USA) equipped
with pressure lock. The methane and carbon-dioxide content were
analyzed by a gas chromatograph (PerkinElmer AutoSystem,
USA). The carrier gas was nitrogen and the temperature of the oven
was maintained at 60 C. Data treatment was carried out according
to a previous publication (Hansen et al., 2004).
The cellulose, hemicellulose, ash and lignin content of CW were
measured as described previously (Ververis et al., 2007). Pectin
content was extracted by alkaline hydrolysis at 95 C for 1 h and
precipitated by adding ethanol (Ranganna, 1987). Protein content
was measured according to the Kjeldahl method.

perature in the linear and quadratic forms and time in quadratic


form are signicant (p = 0.00) for the yields of sugars. The maximum sugar yield of about 41% can be obtained by conducting the
hydrolysis experiment for 6 min reaction time at 150 C. Therefore,
hydrolysis at 150 C and 6 min residence time was used for the rest
of the experiments including limonene analysis, pectin recovery,
fermentation and digestion. The conversions of hemicelluloses
and cellulose to their monomer sugars were 69.48 0.90 and
49.11 0.75%, respectively. No pectin was converted to galacturonic acid and the carbon balance value for hydrolysis reactor input and output was 0.99 0.01.
3.2. Limonene content of the hydrolyzate
Hydrolysis at 150 C for 6 min by dilute-acid followed by explosive pressure reduction (ashing) resulted in drastic decrease of
limonene in the hydrolyzates. The limonene content of hydrolyzate
was 0.0035% w/v. Considering dilution of the slurry of CW during
the hydrolysis by 65.30% due to steam condensation, it can be calculated that 99% of the limonene content of CW was removed
through the ashing in the ash drum. Limonene removed can
be recovered by condensation of vapor outlet of the ash drum.
Concentration of limonene left in hydrolyzate (0.0035% w/v) is
lower than the minimum inhibitory concentration of limonene of
0.01% w/v reported for S. cerevisiae (Winniczuk and Parish, 1997).
3.3. Pectin recovery
Dilute-acid hydrolysis can be considered the rst step in a
method for pectin recovery. The hydrolysis at 150 C for 6 min resulted in solubilization of 83.5% of pectin present in CW, while still
16.5% of the pectin remained in the solid part of the hydrolyzate.
This high solubilization is due to the applied high temperature
and the low pH during the hydrolysis (Aravantinoszaris and Oreopoulou, 1992). Precipitation of pectin content of the hydrolyzate
liquid resulted in recovery of pectin by a total of 77.6% of pectin
content of CWs. The degree of esterication and the ash content
of recovered pectin were 63.7 (0.98) and 4.23 (0.08)%,
respectively.

3. Results and discussion

3.4. Fermentation of CW hydrolyzate to ethanol

3.1. Dilute-acid hydrolysis

The hydrolyzed CWs was supplemented with nutrients and fermented anaerobically by bakers yeast. The concentration of different sugars prior to inoculation was 15.17, 10.88, 2.91 and 4.01 g/l
for glucose, fructose, galactose and arabinose, respectively. The
yeast strain was not able to ferment arabinose, but it could assimilate the hexoses. The fermentation was completed in 24 h, in
which all the fermentable sugars were consumed and ethanol
was produced. Ethanol yield based on total fermentable sugar consumption was 0.43 0.02 g/g. Glycerol, biomass and succinic acid
were the identied by-products, which had yields of 0.10 0.01,
0.070 0.008 and 0.0060 0.0004 g/g, respectively. The carbon
balance in the fermentation was 1.02 0.03.

The citrus wastes from orange juice production were hydrolyzed with 0.5% v/v sulfuric acid at 130170 C for 39 min, and
the results are summarized in Table 1. The maximum sugar yield,
42.05%, was achieved at 150 C and 6 min. However, the more realistic value is the average of results of the 9th to 13th experiments
(Table 1), with a sugar yield value of 41.33 0.56%. Increasing the
temperature and time to more than their optimal values results in
a decrease of the total liberated sugars (Table. 1). This is most likely
due to decomposition of hexose sugars (mainly fructose) to
hydroxymethylfurfural (Grohmann et al., 1995; Talebnia et al.,
2008). A second order mathematical model was tted to obtained
sugar yields:
2

3.5. Anaerobic digestion of CW hydrolyzate to biogas

YTS 418:75 5:66T 7:57t  0:018T  0:37t  0:019tT


1
where t and T are time (min) and temperature (C), respectively.
There is good agreement between values predicted by Eq. (1)
and actual values of sugar yield (Table 1). The tness of the model
was checked by regression coefcient (R2), which was 0.97. It
means that only 3% of the total variations are not explained by
the model. Considering the coefcients of operating variables, tem-

The substrate for digestion had TS and VS contents of 4.6% and


4.3%, respectively. The cumulative methane yield was
0.280 0.01 l/g VS after 10 days and reached a constant level of
0.363 0.02 l/g VS after 30 days. It results in a carbon balance of
1.04 0.03 for the digestion. More than 90% of the maximum produced methane was achieved between 15 and 20 days. Compositions of methane and carbon dioxide in the produced biogas
were 41% and 59% (v/v), respectively. The ultimate yield of meth-

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M. Pourbafrani et al. / Bioresource Technology 101 (2010) 42464250

Citrus Waste

Sulfuric Acid

Water

Hydrolysis Reactor

Steam

Expansion Tank

Condenser/Decanter

Limonene

Hydrolysate
Solid

Liquid
Filter

Biogas Digester

Biogas

Precipitator

Pectin Depleted
Residue

Fermenter

Pectin

Dryer

Dried Pectin

Ethanol

Distillation

Stillage
Fig. 1. Block ow diagram for production of ethanol, biogas, pectin and limonene from CW.

ane can be compared with the value of 0.4550.486 l/g VS reported


by Gunaseelan (2004).
3.6. Overall process
A method for commercial treatment of CW to yield different value-added products including ethanol, biogas, limonene and pectin
is presented in this paper. A simplied block ow diagram of the
process is shown in Fig. 1. By applying this process, 39.64 l ethanol,
almost 45 m3 pure methane, 8.9 l limonene, and up to maximum
38.8 kg pectin can be produced per ton of the wet CW. It is an integrated process, in which the ethanol produced in the process can
be used for pectin recovery, and the produced methane can be utilized in a steam boiler to generate steam required for distillation
and hydrolysis (Fig. 1).
4. Conclusion
In this work, a new process is presented to produce ethanol,
biogas and limonene from CWs. Depending on the market and

protability of the process, pectin can be recovered as a by-product


from the process. Simplicity of the process and low price of biomass compared to other ethanol processes from lignocelluloses
make this process unique and favorable. However, further economic optimizations are required to investigate the protability
of the process.
Acknowledgements
The authors are grateful to the Foundation of Swedbank in
Sjuhrad, Brmhults Juice AB and Sjuhrad Association of Local
Authorities (Sweden) for nancial support of this work.
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