Bioresource Technology 101 (2010) 8993–8999

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

Biomethanization of orange peel waste

M.A. Martín *, J.A. Siles, A.F. Chica, A. Martín
Departamento de Química Inorgánica e Ingeniería Química, Facultad de Ciencias, Universidad de Córdoba, Campus Universitario de Rabanales, Edificio C-3,
Ctra Madrid-Cádiz, km 396, 14071 Córdoba, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Recent research has demonstrated that orange peel waste is a potentially valuable resource that can be
Received 9 March 2010 developed into high value products such as methane. Following a pre-treatment to extract D-limonene,
Received in revised form 23 June 2010 the anaerobic digestion of orange peel waste was evaluated at laboratory and pilot scale under meso-
Accepted 26 June 2010
philic and thermophilic conditions. D-Limonene removals of 70% were reached with pre-treatment. The
Available online 23 July 2010
results showed the convenience of thermophilic conditions for treating this waste as the methane pro-
duction rate and biodegradability were higher than at mesophilic temperature. At pilot scale, a thermo-
Keywords:
philic continuously stirred-tank reactor working in semi-continuous mode was employed. The OLR was
Orange peel waste
D-Limonene
found to be in the range of 1.20–3.67 kg COD/m3 d; the most appropriate range for working under stable
Anaerobic digestion conditions at SRT of 25 d. The methane yield coefficient was found to be 0.27–0.29 LSTP CH4/g added COD
Thermophilic and mesophilic conditions and the biodegradability 84–90% under these conditions. However, acidification occurred at the highest
Biodegradability OLR.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Orange juice is one of the most widely-consumed beverages
today. Consequently, the cultivation of oranges has become a
major industry and an important economic sector in the United
States (Florida and California), Brazil, Mexico, Pakistan, China,
India, Iran, and most Mediterranean countries. According to the
Statistical Database of the Food and Agriculture Organization of
the United Nations (FAOSTAT), world orange production in
2007 was estimated to be 63,906,094 tons. A high percentage
of this production (70%) is used to manufacture products such
as juice or marmalade. Moreover, approximately 50–60% of the
processed fruit is transformed into citrus peel waste, which is
composed of the peel, seeds and membrane residues (Wilkins
et al., 2007a). In order to prevent problems related to the dis-
posal of this product and environmental concerns, this waste
must be properly processed. Under current environmental legis-
lation, all waste must be considered raw material if a valoriza-
tion procedure is to be developed (Möller et al., 2001).
Although orange peel waste can be reutilized for a wide variety
of purposes, until relatively recently there has been no satisfac-
tory means of disposal other than dumping the waste on land
adjacent to production sites, utilizing it as raw material in the
manufacture of cattle feed or burning it (Lapuerta et al., 2008).
However, these processes generate highly polluted wastewater
* Corresponding author. Tel.: +34 957 218586; fax: +34 957 218625.
E-mail address: iq2masam@uco.es (M.A. Martín).
given that the previous pressing stage requires the addition of
binder (Martín et al., 2010). A vast number of promising propos-
als for using this waste have been described in the literature
(Siles et al., 2010). Among others, these include using the waste
to produce fertilizer, essential oils, pectin, ethanol, industrial
enzymes, single cell proteins, pollutant absorbents and paper
pulp supplement. Anaerobic digestion, in which both pollution
control and energy recovery can be achieved, is another possible
way to treat and revalorize abundant orange peel waste. This
process is defined as the biological conversion of organic material
to a variety of end products including ‘biogas’ whose main con-
stituents are methane (65–70%) and carbon dioxide (Wheatley,
1990). The advantages of anaerobic digestion include low levels
of biological sludge, low nutrient requirements, high efficiency
and the production of methane, which can be used as an energy
source for on-site heating and electricity (Nallathambi, 2009).
The activity of the bacteria involved in the process varies with
its age, morphology and temperature, with optimal temperature
conditions at mesophilic (35 °C) and thermophilic (55 °C) range
(Demeyer et al., 1981). Nevertheless, a wide variety of inhibitory
substances are the primary cause of the upset or failure of ther-
mophilic anaerobic digesters (Chen et al., 2008). These include
ammonia, sulfide, light metal ions, heavy metals, and organics
such as phenols (Sierra-Alvarez and Lettinga, 1991), alcohol
(Demirer and Speece, 1998), aldehydes (Gonzalez-Gil et al.,
2002), ether and cetones (Playne and Smith, 1983), and carbox-
ylic acids (Blum and Speece, 1991). Co-digestion with other
waste, the adaptation of microorganisms to these substances

0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.06.133
8994 M.A. Martín et al. / Bioresource Technology 101 (2010) 8993–8999
Nomenclature
Alk alkalinity (mg CaCO3/L)
COD chemical oxygen demand (mg/L)
C2 acetic acid (meq)
C3 propionic acid (meq)
MS total mineral solids (mg/L, %)
MSS mineral suspended solids (mg/L)
OLR organic loading rate (g COD/g VSS
d)
SRT solid retention time (d)
STP standard temperature and pressure conditions (0 °C, 1
atm)
and the use of methods to remove or counteract toxicants before
anaerobic digestion can significantly improve the waste treat-
ment efficiency.
Citrus peel contains essential oils which are well-known anti-
microbial agents (Braddock et al., 1986; Plessas et al., 2007). Essen-
tial oils are contained in oil sacs or glands that range in diameter
from 0.4 to 0.6 mm. The sacs or glands are located at irregular
depths in the flavedo, which is located in the outer rind of the fruit.
Orange peel typically contains 5.436 kg of oil per 1000 kg of or-
anges, of which approximately 90% is D-limonene (Braddock
et al., 1986; Hull et al., 1953); a hydrocarbon classified as a cyclic
terpene. As the main odorous constituent of citrus fruit (plant fam-
ily Rutaceae), D-limonene is employed in the manufacture of food
and medicines as a flavoring agent and has many applications in
the chemical industry as well as in cosmetics and domestic house-
hold products (Smyth and Lambert, 1998). Given these features, it
is essential to extract D-limonene from the peel for anaerobic
digestion processes to take place. Cold pressing, steam distillation
or solvent extraction with hexane or carbon dioxide are relatively
simple and well-established processes of extracting D-limonene
from orange peel (Hull et al., 1953).
The anaerobic digestion of orange peel waste after a pre-treat-
ment makes the addition of binder unnecessary, while preventing
the generation of polluted wastewater. Moreover, the digestate
might be used as a high quality agricultural fertilizer (Tsagarakis
and Georgantzís, 2003).
Our research could contribute to develop a biorefinery ap-
proach. A biorefinery is a facility that integrates biomass conver-
sion processes and equipment to produce fuels, power, and
chemicals ideally from waste biomass. By producing multiple
products, a biorefinery can take advantage of the differences in
biomass components and intermediates, and maximize the value
derived from the biomass feedstock, while producing little waste
(Siles et al., 2010). However, there is little information in the
literature on the digestion of orange waste. Kaparaju and Rintala
(2006) studied the thermophilic anaerobic digestion of industrial
orange waste (pulp and peel) at laboratory scale. In anaerobic
batch cultures, they obtained methane production rates of about
0.49 m3/kg volatile solids (VS) (added peel waste). In semi-con-
tinuous anaerobic cultures, the loading of 2.8 kg VS/m3 d and
hydraulic retention times (HRT) of 26 days generated a specific
methane yield of 0.60 m3/kg VS. However, this required adjust-
ing pH (from an initial 3.2 to 8.0) by adding CaCO3.
The aim of this study was to assess the viability of the anaer-
obic digestion of orange peel waste derived from orange juice
manufacturing under mesophilic and thermophilic conditions
after a pre-treatment to extract D-limonene without adding bin-
der and neutralizing pH. The methane yield coefficient (Y CH4 =S ),
organic loading rate and biodegradability were determined at pilot
scale using a continuous stirred-tank reactor (CSTR) operating in
semi-continuous mode and under thermophilic conditions.
TS total solids (mg/L, %)
TSS total suspended solids (mg/L)
VA volatile acidity (mg acetic/L)
VS volatile solids (mg/L, %)
VSS volatile suspended solids (mg/L)
Y CH4 =S methane yield coefficient (mLSTP CH4/g added VS or LSTP
CH4/g COD added)
2. Methods
2.1. Experimental set-up
The experimental set-up used at laboratory scale for the anaer-
obic digestion of orange peel waste derived from orange juice man-
ufacturing consisted of two 3.5-l continuous stirred-tank reactors
(CSTR) with four connections to load feedstock, ventilate the bio-
gas, inject inert gas (nitrogen) to maintain the anaerobic conditions
and remove effluent. The reactor content was stirred using a stir-
ring blade connected to an engine. Temperature was maintained
by means of a thermostatic jacket containing glycerol at 37 °C for
mesophilic experiments and 67 °C for thermophilic assays. All of
the experiments were carried out in batch mode. The volume of
methane produced during the process was measured using 2-l
Boyle–Mariotte reservoirs connected to each reactor. To remove
the CO2 produced during the process, tightly closed bubblers con-
taining a NaOH solution (6 N) were connected between the two
elements. The methane volume displaced an equal measurable vol-
ume of water from the reservoir.
At pilot scale, the experimental set-up consisted of one 3200-l
CSTR. The temperature of the CSTR (55 °C) was maintained by
means of an electrical thermostatic jacket. The top of the CSTR
was fitted with a tube through which the biogas was transported
to a condenser to remove the moisture. The biogas was then quan-
tified using a flow meter. The reactor was fed in semi-continuous
mode (three times a day). After storing the digestate in a buffer
tank, a centrifuge was used to separate the solid phase from the li-
quid phase. An additional tank was used to chop and store the sub-
strate prior to the anaerobic treatment.
For the mesophilic experiments, the reactors were inoculated
with methanogenically-active granular biomass obtained from a
full-scale anaerobic reactor used to treat brewery wastewater
belonging to the Heineken S.A. Factory (Jaen, Spain). The ther-
mophilic experiments were carried out by inoculating biomass
from a full-scale anaerobic reactor used to treat vegetable and
agricultural wastes from the Colsen b.v. Agency (Hulst, The
Netherlands). Table 1 shows the analytical characterization of
both types of sludge, which were selected on the basis of their
high methanogenic activity (Field et al., 1988). The sludge
showed values ranging from 0.87 to 0.99 g COD/g VSS d for mes-
ophilic bacteria and 0.98–1.09 g COD/g VSS d for thermophilic
microorganisms.
2.2. Substrate
The orange peel used in this study was derived from orange
juice manufacturing processes carried out at the Cítricos del Andé-
valo Company located in Huelva (Spain), which generates 400 tons
of this waste per day. In order to homogenize the orange peel and
 M.A. Martín et al. / Bioresource Technology 101 (2010) 8993–8999 8995

Table 1 is equivalent to 1.361 g ThOD/g orange peel and a higher caloric

Characterization of thermophilic and mesophilic sludge (mean value ± standard power of 4545 kcal/kg.
deviation).

Parameter Thermophilic sludge Mesophilic sludge 2.3. Substrate pre-treatment

pH 7.39 ± 0.02 7.22 ± 0.03
Volatile acidity (mg acetic acid/L) 1774 ± 85 1698 ± 48 The substrate was previously treated by steam distillation in or-
Alkalinity (mg CaCO3/L) 11,607 ± 62 6715 ± 105 der to reduce D-limonene content. The experimental set-up em-
TSS (mg/L) 66,185 ± 838 66,533 ± 720
MSS (mg/L) 20,327 ± 78 11,317 ± 55
ployed for this purpose consisted of an electric heater (520 W,
VSS (mg/L) 45,859 ± 761 55,216 ± 665 200 cm2 contact surface), a 2-l round-bottom flask, a condenser
TS (mg/L) 67,883 ± 794 78,803 ± 805 and a 1-l flat-bottom flask to collect the distillate. Distillation time
MS (mg/L) 19,450 ± 964 13,504 ± 98 varied from 0 to 6 h. The ratio of water to chopped orange peel
VS (mg/L) 48,433 ± 983 65,299 ± 707
waste was fixed at 6:1 (w/w). Distillate samples (25 mL) were ta-
ken and analyzed across distillation times in order to determine
the most appropriate pre-treatment time. Moreover, to determine
improve anaerobic digestion, the peel was chopped using a com- the maximum D-limonene removal yield achieved with pre-treat-
mercial blender until obtaining a final particle size of <2 mm. After ment, the concentration of D-limonene in the raw and pre-treated
chopping, the peel was steam distilled. The aim of this step was not orange peel waste was determined by soxlet extraction for 2 h
to obtain D-limonene, but to reduce the concentration of this com- using 2-propanol as an extractant. Determination in the liquid
pound in order to ensure the stability of the anaerobic process. The phase was carried out according to the method suggested by Scott
final concentration of D-limonene in the influent, which was di- and Veldhuis (1966).
luted to feed the reactor until reaching a final concentration in
the range of 60,000–140,000 mg COD/L, was found to be lower 2.4. Anaerobic digesters: experimental procedure
than 2 mg/L.
Table 2 shows the chemical composition of orange peel before After loading the 3.5-l anaerobic reactors with 12 g VSS/L of
and after steam distillation. For anaerobic digestion to occur, the granular sludge as inoculum, the mesophilic anaerobic digestion
waste must contain carbon, nutrients and oligoelements to main- of pre-treated orange peel waste was studied. The same VSS con-
tain growth and cellular metabolism. The COD:N:P ratio in the centration was used to conduct the thermophilic experiments at
influent varied across the experiments due to variations in the laboratory scale. In all cases, the nutrient and trace element solu-
composition of the processed oranges. However, since the ratio tions described by Fannin (1987) and Field et al. (1988) were added
was always higher than 300:5:1, it was not necessary to add nitro- when the sludge was loaded. Both solutions are very important for
gen and phosphorous for an efficient rapid start-up (Aiyuk et al., activating bacterial growth and metabolism at the beginning of the
2004; Amatya, 1996). This constitutes an economic advantage over process and to compensate for the shortage of nutrients in the
other substrates with a shortage of nutrients. Additionally, differ- substrates.
ent metal concentrations including Cu, Ni, Zn, Cr, Cd and Pb were In order to activate the biomass prior to the experiments, the
determined as the presence of these metals is very important at reactors were first fed with a synthetic solution composed of glu-
trace concentration to ensure the stability of the anaerobic process. cose, sodium acetate and lactic acid (GAL solution) at concentra-
On the other hand, according to the elemental analysis of the waste tions of 50 g/L, 25 g/L and 20.8 mL/L, respectively. During this
at different moments during the production period, the mean initial period, the organic load added to the reactors was gradually
molecular formula of the waste was found to be C44H62O28N, which increased from 0.25 to 1.00 g COD over a 14-day period. After this
previous stage, biomass acclimatization was carried out by increas-
ing the added load with pre-treated orange peel waste to 2 g VS/L
Table 2 over a 16-day period. During this acclimatization period, the vol-
Chemical composition of orange peel waste before and after steam distillation (mean ume of methane was measured as a function of time. Once this pre-
value ± standard deviation).
liminary acclimatization step was completed, a series of batch
Parameter Orange peel Pre-treated mesophilic and thermophilic experiments were carried out using
waste orange peel the pre-treated orange peel at a concentration of 2 g VS/L. Each as-
waste
say lasted a maximum of 120 h; the time interval required to com-
Wet basis pH 3.42 ± 0.02 3.41 ± 0.03 pletely biomethanize each load. In all cases, the volume of methane
VA (mg acetic acid/ 1950 ± 27 220 ± 12
was measured as a function of time and samples were taken and
kg aqueous suspension)
Alkalinity (mg CaCO3/ Under range Under range analyzed before and after feeding. Each load was carried out at
kg aqueous suspension) (pH < 4.5) (pH < 4.5) least in duplicate and the results expressed as means.
Moisture (%) 79.83 ± 0.08 91.15 ± 0.01 At pilot scale, the anaerobic digester was initially loaded with
TS (%) 20.17 ± 0.08 8.85 ± 0.01
the inoculum at a concentration of 8% VS, and the thermophilic
MS (%) 0.87 ± 0.03 0.26 ± 0.05
VS (%) 19.31 ± 0.11 8.58 ± 0.05
anaerobic digestion of pre-treated orange peel waste was studied.
The nutrients, trace elements and easily biodegradable sucrose
Dry basis MS (%) 4.31 ± 0.15 2.95 ± 0.57
VS (%) 95.74 ± 0.55 96.95 ± 0.57
from cane molasses were also added to activate the anaerobic bio-
COD (mg O2/g) 1085 ± 55 1177 ± 46 mass. Biomass acclimatization was then carried out over a 30-day
Total N (mg N/g) 12.24 ± 0.56 11.14 ± 0.04 period. To acclimatize and adapt the anaerobic sludge to the sub-
Kjeldahl N (mg N/g) 11.67 ± 0.24 9.66 ± 0.56 strate studied, the organic loading rate (OLR) was slowly increased
Ammonia N (mg N/g) 1.68 ± 0.19 1.88 ± 0.20
from 0.44 to 1.20 kg COD/m3 d. The reactor was fed in semi-contin-
Total P (mg P/g) 1.18 ± 0.03 2.37 ± 0.03
Cu (mg/kg) Under range 13.3 ± 1.4 uous mode three times per day and the hydraulic retention time
Cr (mg/kg) 1.6 ± 0.7 3.0 ± 0.1 was fixed at 25 days. Bouallagui et al. (2003) suggested that the
Ni (mg/kg) 6.1 ± 1.3 6.2 ± 0.3 most appropriate HRT for the anaerobic digestion of fruit and veg-
Cd (mg/kg) 4.9 ± 0.8 5.8 ± 1.0 etables wastes varies in the range of 10–20 days, although this
Pb (mg/kg) Under range 8.7 ± 1.3
Zn (mg/kg) 4.5 ± 0.4 12.4 ± 1.1
should be slightly higher when the wastes are not mixed with
the other substrates to be digested. On the other hand, Srilatha
8996 M.A. Martín et al. / Bioresource Technology 101 (2010) 8993–8999
et al. (1995) suggested an HRT of 25 days for the anaerobic diges-
tion of orange peel waste even after a fermentative pre-treatment
with Aspergillus, Fusarium and Penicillium. Once this preliminary
acclimatization step was finished, a series of semi-continuous
experiments were carried out increasing the OLR from 1.20 to
5.1 kg COD/m3 d at the same SRT and for a 120-day period. In all
cases, the volume of methane was measured as a function of time
and samples were taken and analyzed at different times before and
after feeding. The digestate was centrifuged and the solid phase
was recirculated or removed until reaching the desired solid reten-
tion time (SRT) (25–30 d).
2.5. Chemical analyses
The following parameters were determined in the influent and
effluent of each load: pH, COD total, COD soluble, TS, MS, VS, vola-
tile acidity (VA) and alkalinity (Alk), total nitrogen, Kjeldhal nitro-
gen, nitrogen as ammonia, nitrate and nitrite, total phosphorous
and soluble orthophosphate. All analyses were carried out in accor-
dance with the Standard Methods of the American Public Health
Association (APHA, 1989).
The biogas composition (CO2 and H2S) was determined using a
Dräger X-am 7000 detector, quantifying the methane (CH4) per-
centage by difference. The volatile fatty acids concentration was
analyzed by using a Gas Chromatograph model HP 5890. The metal
content was determined by atomic absorbance spectrophotometry
using a Perkin–Elmer A-Analyst 300 analyzer. D-Limonene was
analyzed by bromated titration according to the method described
by Scott and Veldhuis (1966).
2.6. Software
Sigma-Plot software (version 11.0) was used to design graphs,
perform the statistical analysis and fit the experimental data pre-
sented in this study.
3. Results and discussion
3.1. D-Limonene extraction
Fig. 1 shows the variation of the D-limonene concentration in
the distillate samples (25 mL) taken during the distillation time.
As can be observed, the D-limonene concentration in the distillate
became stable after 1 h, thus suggesting that, from the economical
point of view, this is the most appropriate distillation time for
250
D-Limonene (µL) / 25 mL distillate
200
150
100
50
0
0 1 2 3 4 5 6 7
Time (h)
Fig. 1. Variation of D-limonene concentration in the distillate versus time.
reducing the concentration of D-limonene in orange peel waste.
Consequently, this time was fixed for the experiments at pilot
scale. On the other hand, the D-limonene removal yield achieved
with this pre-treatment for 1 h was found to be 70%. Concretely,
around 12.5% of the water initially added was necessary to achieve
this yield, which is equivalent to an energy requirement of 1.7 kJ/g
wet orange peel waste. These results are in line with those ob-
tained by Wilkins et al. (2007b), who found that pre-treatment
by steam explosion removed 90% of D-limonene in the orange peel.
3.2. Mesophilic and thermophilic digestion viability
Fig. 2 shows the variation of methane yield coefficient from pre-
treated orange peel waste under mesophilic and thermophilic
conditions in batch mode and at laboratory scale. As can be ob-
served, the methane yield coefficient at standard temperature
and pressure conditions (STP) was found to be higher under ther-
mophilic conditions (332 ± 17 mLSTP CH4/g added VS) than at mes-
ophilic temperature (230 ± 16 mLSTP CH4/g added VS). Additionally,
the mean rate was considerably different in both cases, being high-
er under thermophilic conditions (13.28 mLSTP CH4/g added VS h)
than at the lowest temperature (1.92 mLSTP CH4/g added VS h). This
justifies the election of thermophilic temperature as being the
most suitable temperature for the anaerobic treatment of orange
peel waste at pilot scale given that the reactor volume is smaller;
even when taking into account the higher heating costs under
these conditions. Thermophilic digestion seems to be more prom-
ising than mesophilic digestion as it has a high methane produc-
tion rate, high loading potential, shorter HRT and a consequent
reduction of the digester volume, better pathogen and odor control
and weed seed elimination (Kaparaju and Rintala, 2006). Addition-
ally, Converti et al. (1999) suggested that thermophilic bacteria are
able to use carbon sources that are not biodegradable under mes-
ophilic conditions, as well as hydrolysis rate may increase six times
at thermophilic temperature. On the other hand, the energy
requirements are higher for thermophilic digestion (Zabranska
et al., 2002), although in some cases this cost is compensated for
by an increase in the biodegradable organic matter rate and with
it biodegradability (Bouallagui et al., 2004).
3.3. Stability
The stability of the anaerobic process is a crucial factor for
achieving the appropriate energetic valorization of pre-treated or-
ange peel waste given that an unstabilization process may easily
400
350
YCH /S (mLSTP CH4 /g VS)
300
250
200
150
4
100
Mesophilic temperature (35ºC)
50 Thermophilic temperature (55ºC)
0
0 20 40 60 80 100 120
Time (h)
Fig. 2. Evolution of the methane yield coefficient from pre-treated orange peel
under mesophilic and thermophilic conditions.
 M.A. Martín et al. / Bioresource Technology 101 (2010) 8993–8999
take place when working under thermophilic conditions (Chen
et al., 2008). The stability of the process was evaluated based on
the evolution of the pH, alkalinity, volatile acidity (mg C2/L), vola-
tile acidity/alkalinity ratio (VA/Alk) and volatile fatty acids profile
during the anaerobic digestion process. Table 3 shows the variation
of the pH and VA/Alk ratio under thermophilic and mesophilic con-
ditions. As can be seen, the mean value ± standard deviation of pH
under thermophilic conditions was found to be 8.08 ± 0.19 and
8.60 ± 0.60 at mesophilic temperature during the first step. Given
that the feed pH is 3.41, the original pH levels decreased to
7.40 ± 0.20 and 7.72 ± 0.70, respectively, during the set conditions
and to 6.80 ± 0.12 and 6.70 ± 0.20 when the digesters were acidi-
fied. These values remained within the optimal range for methano-
genic bacteria (Fannin, 1987; Wheatley, 1990). On the other hand,
the VA/Alk ratio indicates the buffer capacity of the reactors’ con-
tents against an acidification process. This was always higher
under thermophilic conditions than under mesophilic tempera-
ture. Specifically, this variable reached values of 0.38 ± 0.07, 0.22
± 0.06 and 0.45 ± 0.07 (eq C2/eq CaCO3) during the start-up, set
conditions and acidification steps, respectively, at thermophilic
temperature and 0.19 ± 0.03, 0.17 ± 0.05 and 0.37 ± 0.02 (eq C2/eq
CaCO3) for the same steps under mesophilic conditions. This vari-
able should not be higher than 0.3 in mesophilic digesters,
although it may be higher for thermophilic digestion a fast unsta-
bilization might take place at high values (Nordstedt and Thomas,
1985). Consequently, the stability and methane production rate
suggest that thermophilic conditions seem to be the most appro-
priate choice for revalorizing pre-treated orange peel waste and
evaluating its methane yield coefficient, biodegradability and or-
ganic loading rate.
3.4. Organic loading rate (OLR) and biodegradability
The organic loading rate in this study varied from 0.44 to
5.1 kg COD/m3 d. Acclimatization was carried out during the first
30 days (0.44–1.20 kg COD/m3 d). Subsequently, the OLR was
gradually increased until reaching a value of 3.67 kg COD/m3 d.
The OLR was then maintained at this value in order to avoid an
acidification process. After this step, the OLR was enhanced until
5.1 kg COD/m3 d, where unstabilization was maximum, showing a
marked decrease in pH decrease and an increase in the VA/Alk
ratio. The VA enhance is a result of the accumulation of volatile
fatty acids. Specifically, the organic acids with a concentration
of more than three carbons increased in the digester, leading to
a higher propionic acid/acetic acid ratio with values in the range
of 0.14 ± 0.02 to 1.30 ± 0.15 and 5.00 ± 0.41 eq (C3)/eq (C2) during
the start-up, set conditions and acidification step, respectively.
Inhibition is usually indicated by a decrease in the steady-state
rate of methane gas production and the accumulation of organic
acids (Kroeker et al., 1979). Finally, the OLR was reduced to
3.00–3.50 kg COD/m3 d and the methanegenic activity was
recovered.
In conclusion, the OLR obtained values in the range of 1.20–
3.67 kg COD/m3 d under stable conditions and with a COD removal
Table 3
Variation of pH, VA/Alk ratio and C3/C2 ratio under thermophilic and mesophilic
conditions.
Parameter Thermophilic conditions Mesophilic conditions
pH VA/Alk (eq C2/ pH VA/Alk (eq C2/
eq CaCO3) eq CaCO3)
Start-up 8.08 ± 0.19 0.38 ± 0.07 8.60 ± 0.60 0.19 ± 0.03
Set conditions 7.40 ± 0.20 0.22 ± 0.06 7.72 ± 0.70 0.17 ± 0.05
Acidification 6.80 ± 0.12 0.45 ± 0.07 6.70 ± 0.20 0.37 ± 0.02
8997
of 84–90%, with 4 kg COD/m3 d being the highest value that may be
reached without acidification and the inhibition of methane pro-
duction. When the digester was acidified, the COD removal was
found to be 63%. Biodegradability was calculated considering the
load added to the reactor and the remnant COD in the liquid phase
of the digestate. These values are higher than those shown by
Bouallagui et al. (2003) for mesophilic conditions, where the high-
est biodegradability was found to be 74.4% and the lowest (acidifi-
cation) 58.6%. Moreover, in the previous mesophilic assays carried
out at laboratory scale, the biodegradability of the pre-treated
waste was found to be 65%. After thermophilic anaerobic digestion,
the remnant soluble COD in the liquid phase of the digestate was
still high for spilling and contained high concentrations of nitrogen
and phosphorus. Moreover, given that the orange juice plant pro-
duces wastewater with high biodegradable organic matter content,
but with low nitrogen and phosphorous content, one possibility
could be to unify both wastewaters and treat the mixture in an aer-
obic membrane reactor (MBR). In regions in which the soil requires
nutrients, the mixture of wastewater effluent is suitable to be uti-
lized as liquid fertilizer.
3.5. Methane yield coefficient and biogas composition: thermophilic
conditions
The generation of methane is of special interest as methane is a
useful compound due to its caloric power (lower caloric power):
35,793 kJ/m3, equivalent to 9.96 kW h/m3. Obtaining methane al-
lows low-priced or inconvenient substrates with a high concentra-
tion of organic matter to be revalorized. Consequently, the
composition of the biogas and the methane yield coefficient are
the most interesting factors to be considered from the energetic
point of view.
Fig. 3 shows the evolution of the methane yield coefficient
with the organic loading rate during the thermophilic experi-
ments at pilot scale, including start-up, set conditions and
acidification. During the start-up, the methane yield decreased
from 0.576 to 0.241 LSTP CH4/g added COD when the OLR was
increased from 0.44 to 1.20 kg COD/m3 d. This was probably
due to the fact that the first loads contained additional soluble
organic matter from previous activities or cellular lyses as a
consequence of adapting the substrate. For OLR ranging from
1.20 to 3.67 kg COD/m3 d, the yield remained relatively stable
(around 0.27–0.29 LSTP CH4/g added COD). Finally, for OLR in
the range of 3.67–5.10 kg COD/m3 d this variable decreased to
0.7
0.6
YCH /S (LSTP CH4 /g CODadded)
0.5
0.4
0.3
0.2
4
0.1
START-UP SET CONDITIONS ACIDIFICATION
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
OLR (kg COD/m3·d)
Fig. 3. Variation of the methane yield coefficient with the organic loading rate
(OLR) during the thermophilic experiments at pilot scale.
8998
100
90
Methane percentage in the biogas (%)
80
70
60
50
40
30
20
10
0 bacteria and its use in interspecies comparisons and correlations. Res. J. Water
SET CONDITIONS ACIDIFICATION
Fig. 4. Variation of the methane percentage in the biogas during the experiments.
0.09 LSTP CH4/g added COD as a consequence of the acidification
process and the inhibition of the methanogenic bacteria. These
results are in line with those shown by Lane (1984), who ob-
tained a methane yield of 0.5 m3/kg TS from orange peel (10%
TS) fed semi-continuously at an organic loading rate (OLR) of
3.5 kg TS/m3 d. Nevertheless, methane production can be im-
proved by pre-treating the orange peel with selected strains of
Sporotrichum, Aspergillus, Fusarium and Penicillium (Srilatha
et al., 1995). Like the COD removal yield, the methane composi-
tion in the biogas also varied. When the OLR allowed working in
stable conditions (1.20–3.67 kg COD/m3 d), the methane percent-
age in the biogas reached the highest value, which was found to
be around 55% (Fig. 4). Higher OLR (>3.67 kg COD/m3 d) led to
acidification and a simultaneous decrease in the methane per-
centage of the biogas. Later, when the stability was regained,
the percentage increased again, until reaching a final value of
40%. On the other hand, the H2S concentration was found to
be lower than 200 ppm, making it unnecessary to post-treat
the biogas and permitting it to be directly burnt, with the con-
sequent economic advantage. The rest of the sulfur content in
the substrate remained in the digestate, which could be used
as a fertilizer.
4. Conclusions
Anaerobic digestion of orange peel waste after a D-limonene
extraction reveals higher methane production, methane produc-
tion rate and biodegradability in thermophilic than mesophilic
conditions. The highest methane yield coefficient at pilot scale
and thermophilic temperature was 0.27–0.29 LSTP CH4/g added
COD, obtained at an OLR of 1.20–3.67 kg COD/m3 d. Biodegradabil-
ity was found to be 84–90%, although a strong inhibition process
was observed when the OLR was higher than 4 kg COD/m3 d,
reaching a normal manner again when the OLR was reduced.
Anaerobic digestion provides an excellent opportunity to integrate
this waste into a Biorefinery approach involved in orange juice
manufacturing.
Acknowledgements
The authors are very grateful to the Cítricos del Andévalo S.A.
Company (Huelva, Spain), concretely to José Antonio Maldonado,
Colsen b.v. Agency (Hulst, The Netherlands) and Ahidra S.L. (Barce-
lona, Spain) for funding this research. We also wish to express our
gratitude to the Spanish Ministry of Science and Innovation for
M.A. Martín et al. / Bioresource Technology 101 (2010) 8993–8999
funding José Ángel Siles López through Project CTM2005-01293
and Grant BES-2006-14074. This study was co-financed by the
European Social Fund.
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