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Valorization of Residual Orange Peels: Limonene Recovery, Volatile

Fatty Acids, and Biogas Production
Federico Battista,* Gianmarco Remelli, Serena Zanzoni, and David Bolzonella

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ABSTRACT: The new European Union directives on waste

management encourage the adoption of the “pyramidal hierarchy”
for the extraction or synthesis of high-added-value compounds
rather than the conventional mere biogas production. This
Downloaded via 190.237.1.243 on February 4, 2024 at 17:16:26 (UTC).

approach was applied to orange peels (OPs), rich in limonene, a

molecule used as an additive in the cosmetics market. Soxhlet
extraction was optimized, investigating the effects of different
solvents, operative temperatures, cycle number, and substrate−
solvent ratios on limonene recovery. It was demonstrated that n-
hexane, used at a substrate−solvent ratio of 1:2 at 70 °C, gave the
best performance in limonene extraction yield (about 1.20% w/w
of OPs). The residual OPs after limonene extraction were
fermented in batch conditions for the production of volatile fatty acids (VFAs) at different total solids (TS) concentrations. The
best yield of 0.34 gVFA per gram of TS treated was obtained with a TS concentration of 15% w/w and a fermentation duration of 5
days. Finally, the methane potential of extracted and unextracted OPs was tested through anaerobic digestion. Both the tests
achieved the same level of methane productivity of 355−365 NLCH4/kgTVS, but the OPs without a previous limonene extraction
needed double the time to reach the final methane production.
KEYWORDS: orange peels, biorefinery, limonene, solid−liquid extraction, volatile fatty acids, dark fermentation, biogas,
anaerobic digestion

■ INTRODUCTION
The new environmental policies of the European Union focused
on two main points: (i) the need to strengthen the separate
collection of wastes and (ii) the encouraging the pyramidal
hierarchy for waste valorization (Figure 1). This model aims to
minimize the amount of wastes through the recovery of high-
added-value compounds still present in them and produce
innovative and environmentally friendly products (stage
“recycling” of the pyramidal hierarchy in Figure 1). In this
way, wastes are substrates for a secondary raw materials market
creating new job opportunities and thus economic gain. In the
specific case of organic wastes, they can be also exploited for the
production of biofuels and bioenergy (stage “other recovery” in
Figure 1) but only after the recycling stage. Instead, the landfill
disposal and incineration of wastes should be considered as the
last choice or avoided at all.1
This approach has been applied in this study to evaluate the
potential of one of the most produced agro-industrial organic
wastes worldwide: orange peels (OPs) derived from the
cultivation, processing, and consumption of oranges.

Received: March 2, 2020

Revised: March 27, 2020
Published: April 14, 2020

Figure 1. Pyramidal hierarchy for waste valorization from https://ec.

europa.eu/environment/waste/framework.

© 2020 American Chemical Society https://dx.doi.org/10.1021/acssuschemeng.0c01735

6834 ACS Sustainable Chem. Eng. 2020, 8, 6834−6843
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Figure 2. Main phases of this study.

The annual production of oranges has been estimated to be conducted at different durations (3−7 days) and different total
about 70 million tons at the international level, with the United concentrations (15−25% w/w) to determine the best operating
States, and in particular Florida, leading with 7.5 million tons. In conditions. The cascade biorefinery was closed by the biogas
the European Union, orange cultivation is essentially concen- production by the anaerobic digestion (AD) of OPs in batch
trated in Mediterranean countries such as Italy, Spain, and condition, comparing the methane potentials of OPs before and
Greece, amounting to a total annual production of about 6.0 after the recovery of limonene.
million tons.2 Orange pulp and OPs are wastes from oranges, of
which OPs represent 60−65% of the total orange wastes. OPs
are receiving great attention for their high concentration of
■ EXPERIMENTAL SECTION
OPs and Inoculum Characterizations. OPs were collected from
limonene. This compound is a cyclic monoterpene (C10H16), the cafeteria of the Department of Biotechnology of the University of
presenting two different optical isomers: D-limonene and L- Verona. Their main chemical and physical parameters are summarized
in Table 1.
limonene, each with a different fragrance. The smell of D-
limonene is similar to that of orange, while that of L-limonene is
similar to that of turpentine.3 These characteristics made Table 1. Characteristics of OPs and Inoculum
limonene one of the most used flavoring molecules for OPs inoculum
application in cosmetics with a very high production cost pH 4.56 ± 0.25 8.53 ± 0.14
(8.55 Euro/kg) and a commercial price of about 19 Euro per 5 TS (% w/w) 28.88 ± 1.55 5.86 ± 0.35
mL of product (www.sigmaaldrich.com). Despite its high value, TVS (% TS) 97.48 ± 0.74 59.70 ± 0.35
limonene production is very low and has been estimated to be 70 TotCOD (g/L) 380 ± 45 12.40 ± 0.24
and 4 kg/y at the international level and in European countries, NH4+ (g/L) 0.95 ± 0.20 4.04 ± 0.18
respectively.2 For these reasons, limonene was added to the list, N (mg/g) 10.13 ± 0.37 5.30 ± 0.52
prepared by the European Commission, of the 50 most P (mg/g) 1.13 ± 0.04 1.83 ± 0.21
economically valuable bio-based compounds.3 porosity (%) 41.13 ± 2.49 not determined
The need to recover limonene is also due to its inhibiting total VFAs (mg/L) not determined 146 ± 19
effect on the fermentation process, which drastically reduces the
yield of biofuel production.2 Limonene is usually recovered from
the orange essential oil (OEO) extracted from OPs. The largest The anaerobic inoculum used for the AD assays was derived from the
fraction of volatile compounds (90−95%) in OEO consists of Isola della Scala biogas plant (Verona, Italy) by treating a mixture of
bovine manure, chicken manure, and rice straw and operating under
monoterpenes and sesquiterpene as well as their oxygenated mesophilic conditions (35 °C).
derivatives. Oxygenated compounds (5−10%), such as Description of the Unit Operations Involved in the
aldehydes (citral), alcohols, and esters, confer organoleptic Biorefinery for OP Valorization. The OPs, collected from the
properties to the fruit. The concentration of limonene in OEO is cafeteria of the department, underwent different chemicophysical and
about 90%.2 biological processes to follow the cascade biorefinery approach
This study created a lab-scale “cascade biorefinery” model, reported in Figure 2.
based on innovative sequential unit operations for the The OPs were reduced to particles of size 2 mm and kept at 60 °C for
3 days to evaporate the water content. The choice of the particle size
exploitation of OPs (Figure 2). In particular, the solid/liquid was made based on some preliminary tests, the results of which have not
extraction by polar and nonpolar solvents was tested at different been reported or published. In particular, we loaded 5 g of OPs with
temperatures, number of cycles, and substrate−solvent ratios dimensions in the range of 10−1 mm in different beakers filled with 100
using a Soxhlet apparatus to optimize the recovery of OEO from mL of n-hexane, one of the most promising solvents according to the
OPs. Then, the OEO was analyzed by H NMR and mass literature. The tests were conducted under mixing at 100 rpm for 3 h.
spectrometry (MS) techniques to detect and quantify the Then, the orange peels were removed by filtration, while the liquid
limonene concentration. The extracted OPs were further used phase was recovered and weighed after solvent evaporation. It is well
known that the best oil recovery yield was obtained with particles of size
for the fermentation of volatile fatty acids (VFAs). VFAs are one
2 mm. Below this value (1 mm), the amount of oil recovered was also
of the largely used compounds in the chemical industry that the same. The particle size of 2 mm was also adopted by Ruiz et al.5
serve as starting materials for biofuel production and for the After preliminary tests, 50 g of dried SCG was used for the solid/liquid
synthesis of a variety of products, such as biopolymers, reduced extraction trials in a Soxhlet apparatus (purchased from Exacta +
chemicals, and derivatives.4 Batch fermentation tests were Optech, Italy), testing different polar and nonpolar solvents with the

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aim to recover an OEO rich in limonene. The solvent was removed
from the solvent−oil mixture using a preweighed rotary evaporator flask
(IKA Rotary Evaporators RV3V, Germany). The OEO from the
different solid/liquid extractions was characterized by the NMR
analysis for the detection of limonene. Then, the OEO from the polar
and nonpolar solvents with the highest oil recovery yield was quantified
in an external chemical laboratory in Milan (Italy). It was found that the
best solvent for limonene recovery was n-hexane. Consequently, n-
hexane extraction was optimized by testing at different operating
temperatures, substrate−solvents ratios, and number of cycles.
The OPs, after limonene recovery by n-hexane, were tested for the
production of VFAs by dark fermentation at a high content of dry
matter (15−25% w/w), at different durations of fermentation trials (3−
7 days). Finally, AD tests were also conducted to estimate the
production of biogas from OPs before and after limonene extraction.
The details of each biological process are provided in the following
paragraphs.
Orange Essential Oil Extraction. A first round of solid/liquid
extraction tests was conducted to individuate the best solvent for
limonene recovery from OPs. A dried SCG sample (50 g) was placed in
a cellulose extraction thimble (Whatman Cat. No. 2800-373) and
plugged with cotton. The thimble was located in a Soxhlet extractor.
Different polar and nonpolar solvents (300 mL; acetone, chloroform,
diethyl ether, ethanol, ethyl acetate, and n-hexane), selected based on
the oil recovery yields obtained by previous works,3,6,7 were tested in
triplicate for the OPs solid/liquid extraction. A 50:50 (v/v) mixture of
n-hexane−ethanol and n-hexane−acetone was also tested considering
its high performance in terms of OEO obtained by previous works
reported in the scientific literature.5,8 All of the solvents were purchased
from Sigma-Aldrich and tested at 85 °C, a value greater than the boiling
point of any of the solvents. The Soxhlet extraction was stopped after
one extraction cycle.
The extracts (OEO−solvent mixture) were transferred to a predried
and preweighed rotatory evaporator flask. The amount of solvent
recovered from the rotatory evaporator flask was measured to evaluate
the solvent consumption in each cycle of the Soxhlet extraction. Then,
the OEO was put in a chemical hood for evaporation of the eventual
solvent traces until it reaches a constant weight. The performances of
the different solvents were evaluated in terms of OEO recovered from
OPs and expressed as OEO yield (OEOY), calculated according to eq 1
Moil
OEOY(% w/w) = × 100
MOPs (1)
where Moil is the mass of the extracted OEO and MOPs is the mass of the
OPs dried sample.
OEO (0.025 mL) from n-hexane and ethanol (nonpolar and polar
solvents with the best OEOY, respectively) extractions was selected for
1
H NMR analysis. OEO from n-hexane was diluted in 1 mL of
chloroform-d (CDCl3), while the ones extracted from ethanol were
diluted in methanol-d (CD3OD).9
NMR spectroscopy analysis of oil samples was carried out at 298° K
using a Bruker Advance III 600 spectrometer (United States), operating
total VFAs produced at the end of the dark fermentation − endogenous VFA (g VFAs)
YVFAs(%) =
amount of the TS referred to the OPs fed at the beginning of each test (gTS of OPs)
The concentrations of the total VFAs and each acid components were
determined according to the methods described below. The VFA
concentrations are expressed in terms of COD.
Biogas Production from OPs. OPs, after (extracted OP tests) and
before (OP tests) limonene recovery by the n-hexane solid/liquid
extraction, were also tested for determining the biogas production using
conventional anaerobic digestion batch trials. It was therefore possible
to estimate if and how much limonene can inhibit the AD process. The
methane yield was evaluated through biomethane potential (BMP)
tests, which were carried out following the methodology suggested by
Angelidaki et al.14 Trials were carried out in 0.5 L reactors, with a 150
mL working volume, sealed with chloro-butyl caps. Each trial was
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at 600.13 MHz, and equipped with a cryoprobe. 1H NMR spectra were
recorded using the Bruker-standard sequence with 8 or 16 scans and a
spectral width of 12 019 Hz. Two-dimensional (2D) 1H−1H total
correlated spectroscopy (TOCSY) spectra were recorded in a mixing
time of 60 ms using Bruker’s “mlevesgpph” pulse sequence. The 2D
spectra were acquired with four transients, a time domain of 2k points
(F2) and 512 experiments (F1), and a spectral window of 10 200 Hz in
both dimensions. All spectra were manually phased and baseline-
corrected using TOPSPIN 3.6.1 (Bruker) and were referenced to TSP
signal at 0 ppm. The 1H NMR signals of identified molecules were
assigned based on comparisons with the chemical shifts of standard
metabolites in the Biological Magnetic Resonance Data Bank.10
NMR analysis has been adopted for the individuation of limonene in
OEO, while its quantification was performed in an external laboratory
that conducted an HPLC analysis based on the GC/MS/FID system.
This analytical technique combines the separation properties of gas
chromatography (GC) with the detection feature of mass spectrometry
(MS) and flame ionization detector (FID) to identify and quantify
different substances within a test sample.
Optimization of the Solid/Liquid Extraction with n-Hexane.
Considering the OEOY and the limonene content in the OEO, n-
hexane was selected as the best solvent for limonene recovery. Thus, the
process was optimized by testing different operative parameters with
the aim to increase the OEOY and, possibly, to reduce the energetic and
environmental impact. The solid/liquid extraction of n-hexane, already
tested at 85 °C, was also performed at 60 and 100 °C. After the
optimization of the operating temperature, different OP−hexane ratios
of 1:6, 1:3, 1:2, and 2:3 (g/mL) were tested to reduce the n-hexane
consumption. Finally, different extraction cycles (from 1 to 3) were
adopted to evaluate the eventual permanence of limonene in the OPs
after the first extraction cycle.
Dark Fermentation for the VFA Production from the
Extracted OPs. The VFA production was performed at a high content
of TS to reduce the water use for dilution, the volume of the reactors,
and, consequently, the costs for a future pilot and full-scale plant.11 In
particular, the OPs were first placed in an oven, provided with an
opportune suction system, at 60 °C to evaporate the solvent. Then, the
OPs were fermented at 15, 20, and 25% w/w TS under the mesophilic
batch condition (35 °C) in a glass bottle with a working volume of 500
mL. Before the addition of different amounts of OPs, the inoculum was
kept idle for 24 h at an acidic pH (pH 3) with the aim to remove
methanogenic microorganisms, which consume the obtained VFAs for
the synthesis of methane. In this way, the presence of VFAs producing
microorganisms that are highly resistant to extremely low pH, can be
assured.12 The inoculum was reported at a neutral value before the
addition of OPs. Blank tests were also conducted to determine the
endogenous VFA production.
The dark fermentation was performed at different durations of 3, 5,
and 7 days, typically adopted for VFA production,13 and at uncontrolled
pH. All of the tests were conducted in triplicate.
The tests were evaluated considering the VFAs yield referred to the
initial TS amount of OPs, according to the following equation
× 100
(2)
performed in triplicate. The duration of the tests was determined
following the protocol by Holliger et al.,15 which established that BMP
tests have to be stopped when the daily biogas production is lower than
1% of the cumulative one for three consecutive days.
The inoculum (Table 1) was incubated at the test temperature (37
°C) for 1 week to achieve the endogenous methane production. Blank
tests were also conducted to determine the endogenous methane
production. Moreover, microcrystalline cellulose BMP tests were used
as a positive control.14−16 All batch digesters were manually stirred once
a day. The volume of biogas generated during the batch trials was
determined by the water displacement method, while the methane
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Figure 3. OEOY performances from orange peels. Yellow indicates the OEO where limonene was detected by NMR analysis.
Figure 4. 1H NMR spectra of essential oils extracted by diethyl ether (green) and n-hexane (red) and the 1H NMR spectrum of limonene for
comparison (blue) (HMDB ID: HMDB0004321).
content was determined using a portable biogas analyzer (Geotech
BIOGAS 5000 by GeoTech, United Kingdom).
Analytical Methods. The pH of the samples and the pH during
different analyses were measured by a benchtop pH meter purchased
from Mettler Toledo (United States). Each substrate was analyzed in
terms of dry matter (TS), volatile solids (TVS), total chemical oxygen
demand (totCOD), total Kjeldahl nitrogen (TKN), and total
phosphorus (TP) according to the Standard Methods.17 In particular,
COD was determined by the dichromate oxidation method and the
samples were digested in a block digester at 150 °C for 2 h (Velp
Scientifica, Italy). TKN and TP were processed by a high-performance
ETHOS One microwave digestion system by Milestone (Italy) and the
UDK 129 distillation unit by Velp Scientifica (Italy). The
concentrations of the total and each VFAs were determined as follows:
The samples were diluted with distilled water in a substrate−water ratio
of 1:100 and filtered at 0.45 μm. The concentrations of the VFAs were
measured by ionic chromatography (Dionex ICS-1100 with AS23
column, Thermo Fisher Scientific, United States).18 The total grade of
porosity of the OPs was evaluated by the N2 adsorption isotherm
method19 in an external laboratory.
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■ RESULTS AND DISCUSSION
Orange Essential Oil Extraction. Different solvents were
used for oil extraction tests. Figure 3 shows the OEOY obtained
from different polar and nonpolar solvents. Limonene was
detected only in the essential oil recovered by n-hexane and
diethyl ether, marked in yellow.
The higher OEOY performances were obtained by polar
solvents ethanol (24.18% w/w) and acetone (12.99% w/w),
which were able to extract from OPs different soluble sugars, in
particular glucose, fructose, and sucrose, but not limonene, as
revealed by the NMR analysis. Moreover, it is interesting to
observe that a mixture of these two solvents with n-hexane
reduced the OEOY performances, from 24.18 to 21.03% w/w
for ethanol and from 12.99 to 10.86% w/w for acetone. On the
contrary, nonpolar solvents, n-hexane and diethyl ether, showed
very low performances in terms of OEOY: 1.31 and 0.77% w/w,
respectively. However, this was not a negative result, as these
values were higher than those for the typical essential oil
recovery from OPs (around 0.50−1.00% w/w) through the
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Soxhlet apparatus.20 Moreover, the most relevant result was that
the NMR analysis detected limonene in these OEO (Figure 4).
In particular, NMR spectra showed that higher concentrations of
limonene were found in the OEO recovered with n-hexane,
whose spectra are very similar to the limonene ones, than with
diethyl ether.
Different fatty acids (not identified) were also present in the
OEO from n-hexane and diethyl ether extractions.
The capacity of solvents to recover limonene depended on the
chemical affinity between the solvents and limonene, which
influenced its solubility and, consequently, the solid/liquid
extraction yield. The solvent−limonene chemical interaction
can be expressed by the water−octanol partition coefficient
(Kow), which is defined as the ratio of the concentrations of a
solute between two solvents: a hydrophobic solvent (octanol)
and a hydrophilic one (water). Consequently, Kow expresses a
measure of lipophilicity or hydrophobicity of the compounds.21
Negative Kow values are typical of very hydrophilic molecules,
while positive Kow values denote lipophilic compounds. Kow
values close to zero demonstrate the double nature (hydrophilic
and lipophilic) of the solvent.22
Limonene has a very high Kow of 4.2, which reveals its high
lipophilic nature. Consequently, its absence in the OEO from
ethanol and acetone is justified by the hydrophilic nature of the
solvents. Their high OEOY values are explainable by their
tendency to extract soluble sugars, which are known to be
hydrophilic molecules and are present in high concentrations in
OPs (more than 30 w/w).23
Instead, the strong affinity between limonene and n-hexane,
the solvent that showed the highest recovery of limonene, is
justified by the very similar Kow values (Table 2). Limonene was
Table 2. Parameters Influencing the OEOY Performances of
the Different Solvents24
Kow P (polarity boiling solvent
solvents (log P) index) temperature (°C) recovery (%)
acetone −0.24 5.1 56.0 67.01 ± 4.63
chloroform 1.97 4.1 61.2 75.33 ± 3.98
diethyl ether 0.83 2.8 34.6 60.16 ± 5.53
ethanol −0.31 4.3 78.4 82.15 ± 2.41
ethyl acetate 0.73 4.4 77.1 80.00 ± 1.20
n-hexane 3.60 0.1 68.0 77.78 ± 1.34
also found in OEO extracted by diethyl ether, in a small amount.
The lower concentration is consistent with the diethyl ether
Kow, having a positive value, which reveals its lipophilic
behavior, even if it was not so marked as for n-hexane. The Kow
of chloroform is not coherent with the absence of limonene in
the relative OEO. This result can be justified by adopting
another parameter, the polarity index (Table 2). Acetone, ethyl
acetate, ethanol, and chloroform all have high polarity indices of
5.1, 4.4, 4.3, and 4.1, respectively, which make them not
compatible with limonene, a nonpolar compound.25 n-Hexane
has a polar index very close to zero, revealing its compatibility
with the nonpolar nature of limonene and, thus, with its ability to
extract a high amount of it from OPs. Diethyl ether has an
intermediate polarity index, which justifies the presence of
limonene in the corresponding OEO, even if a small amount.
The validity of n-hexane as an extranet agent is also supported by
previous research works. Ozturk et al.25 tested different solvents
to extract limonene from OP and found that the best one was
cyclopentyl methyl ether, which reached an OEOY of about
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1.65% w/w, working at 70 °C and with a solid/liquid ratio of
1:10. This performance is very close to that of OEOY obtained
for n-hexane in this work, equal to 1.31% at 85 °C, with a more
convenient solid/liquid ratio of 1:6. Considering the higher cost
of cyclopentyl methyl ether than n-hexane (respectively, 186
Euro and 127 Euro per liter (www.sigmaaldrich.com)), n-
hexane represents a more convenient choice as a solvent for
limonene extraction. Moreover, the process can be further
improved by the optimization of the operating parameters, as
commented below. A previous work, conducted by Wikandari et
al.,26 tested different solvents (n-hexane, diethyl ether, dichloro-
methane, and ethyl acetate) for limonene recovery before the
anaerobic digestion for biogas production from OPs. Even
though their work did not declare the OEOY from OPs, they
confirmed that a higher limonene recovery was achieved
adopting n-hexane. A unusually high OEOY was achieved by a
recent work,27 where OEO was performed by different drying
processes of OPs. In particular, freeze drying achieved an OEOY
of about 7% v/w (limonene concentration of 80% in the OEO),
corresponding to about 5.9−6% w/w, considering the density of
OEO of about 0.85 g/mL. This high value can be probably
explained by a more efficient water removal from OPs, which
allows a better diffusion of the solvent through the pores of OPs.
Moreover, it is important to remark that the work by
Farahmandfar et al.27 is the only one reporting this very high
OEOY.
Regarding the characterization and quantification of the OEO
from n-hexane, D-limonene was the main constituent accounting
for 92.39% w/w, followed by β myrcene, with 2.84% w/w. This
result is consistent with the ones from previous works.27,28 The
HPLC analysis was also performed on OEO from ethanol
extraction, a polar solvent with the highest OEOY (Figure 3).
Limonene was detected only at a very low percentage, 0.3% w/
w. Previous research works showed that the main constituents of
OEO from ethanol are soluble sugars (glucose, fructose, and
sucrose), representing more than the 80% w/w of OEO.6,8 In the
light of our biorefinery scheme, the extraction of these
compounds is not convenient as they are the main substrates
for the following unit operations on OPs: dark fermentation and
anaerobic digestion, respectively, for the production of VFA and
biogas.
Finally, solvent recovery is a very important factor to be
considered in the economic assessment of the process. The
analysis of Table 2 shows the existence of a correlation of the
boiling point of the solvents and their degree of recovery: the
highest solvent recovery was achieved by ethanol (82.15%), the
solvent with the highest boiling temperature and thus a lower
volatility. However, lower solvent recoveries were observed with
diethyl ether (60.16%) and acetone (67.01%), presenting the
lowest boiling temperatures among the solvents adopted in the
present work. Between them, there was n-hexane, the solvent
with the highest limonene extraction yield, which presents a
good solvent recovery of almost 78%.
Optimization of the Solid/Liquid Extraction with n-
Hexane. Considering the OEOY and the limonene concen-
tration, n-hexane was selected as the best solvent for limonene
recovery from OPs. Thus, the solid/liquid extraction with this
solvent was optimized considering different ranges of parame-
ters: (i) operative temperature, (ii) number of cycles, and (iii)
OP−n-hexane (g/mL) ratio.
In the first stage, the solvents were all tested at 85 °C. n-
Hexane was also tested at lower (70 °C) and higher (100 °C)
temperatures, evaluating the difference in terms of the OEOY.
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Figure 5. Effect of temperature on the OEOY and extraction duration.
Figure 6. Effect of n-hexane−OP ratio on OEOY.
Figure 7. VFA yields and profile from the dark fermentation of OPs.
The OEOY was inversely proportional to temperature: at 60
°C, the OEOY was about 1.70% w/w; at 85 °C, it was 1.31% w/
w, and it decreased to 0.28% w/w at 100 °C. It is also interesting
to observe that temperature had a strong impact also on the
duration of the extraction cycle (Figure 5). In fact, the lower the
operating temperature, the slower the solvent evaporation and
the higher the duration of the extraction cycle. In this way, when
the operating temperature was low (60 °C), the solvent spent
more time in contact with OPs in the Soxhlet extraction
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chamber, improving the OEOY. On the contrary, a high
operating temperature (100 °C) allowed a very quick n-hexane
evaporation and, consequently, a lower contact time with
OPs,21,29 which also significantly reduced the OEOY. Taking
these results into account, an operating temperature of 60 °C
cannot be selected for the optimization of the limonene
recovery, considering the long duration of the extraction cycle
(4.25 h). However, an operating temperature of 85 °C can be
assumed a good compromise between a good OEOY perform-
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ance (1.30%), not as high as the one recorded at 60 °C, and the
duration of the extraction cycle of 45 min.
The optimal number of extraction cycles was also investigated
to verify if OEO can be recovered in just one cycle or if some
residual essential oil remained in the OPs, requiring multiple
passages in n-hexane. It was observed that the increase of the
extraction cycles (from 1 to 5) did not correspond to a
significant variation of OEOY, which remained around 1.25−
1.30% w/w. It demonstrates that n-hexane is very selective and
chemically compatible with limonene and that one extraction
cycle is enough to recover it completely. This result confirms the
work by Xhaxhiu et al.,8 who observed a slight decrease in
limonene recovery with the augmentation of the Soxhlet cycle
number.
Finally, in the solid/liquid extraction process, the substrate−
solvent ratio is one of the most important factors affecting the
process from the economic point of view. The selection of the
best solvent in the previous stage of this work was conducted by
adopting a substrate−solvent ratio of 1:6. The following
different ratios were also tested to find the maximum amount
of OPs, which did not reduce the OEOY. Figure 6 shows that the
amount of OPs can be increased up to a substrate−solvent ratio
of 1:2 without decreasing the OEOY, which remained around
1.30% w/w. Then, further augmentation of OPs started to be not
adequately extracted by the same volume of n-hexane. This can
be probably explained by the reduction of the transfer of solvents
through the pores of OPs,30 as an effect of the lower contact
surface between n-hexane and the OP particles, too pressed at
that condition. Thus, the maximum OP−n-hexane ratio that
resulted in a high OEOY was 1:2.
Dark Fermentation for the VFA Production from the
Extracted OPs. After achieving OEO by n-hexane solid/liquid
extraction, batch trials of dark fermentation for the VFA
production were performed at different TS concentrations (15−
25% w/w), different durations (3−7 days), and uncontrolled
pH.
Figure 7 summarizes the VFA yields at different TS
concentrations and durations of the OP fermentation tests.
Moreover, the percentages of the produced VFAs at different
durations were summarized for the considered TS concen-
trations of 15 and 20% w/w. Instead, considering the inhibition
occurred to the dark fermentation, the VFA profile correspond-
ing to the TS concentration of 25% w/w was omitted.
The best performances were achieved by the dark
fermentation of OPs performed at 15% TS w/w: around 22,
34, and 32% (corresponding to 3.7, 5.7, and 5.5 g of total VFAs/
L) after 3, 5, and 7 days of fermentation, respectively. The
VFA(COD)/TotCOD values were about 18, 29, and 28% after 3,
5, and 7 days, respectively. The increase of the TS concentration
to 20% w/w resulted in the inhibition of the dark fermentation
with VFA yields of around 4, 5, and 26% (corresponding to 1.7,
2.1, and 10.8 g of total VFAs/L) after 3, 5, and 7 days,
respectively. The VFA(COD)/TotCOD values were about 4.6,
5.6, and 29% after 3, 5, and 7 days, respectively. Finally, the
process was completely intoxicated at the TS concentration of
25% w/w, with very low VFA performances: lower than 3%
(between 0.5 and 1.0 g total VFA/L) for all of the considered
durations of the dark fermentation. Consequently, the
VFA(COD)/TotCOD values were around 0.3−1.3%, demonstrat-
ing the inhibition of the system.
VFA production by dark fermentation is generally favored by
short durations, usually 1−7 days according to the chemical and
physical natures of the substrates, and a high organic matter
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concentration.13 This allows us to explain the very good YVFAs
obtained at a TS concentration of 15% w/w at short durations
(3−5 days). Different authors explained that these conditions
favor the glycolytic pathway allowing the VFAs production,
inhibiting the methanogens. On the contrary, longer durations
of the tests enable the activity of microorganisms producing
hydrogen or methane from VFAs.31 It was confirmed by the
YVFAs after 7 days, which started to decline, from 34 to 32%,
meaning that hydrogen-producing microorganisms and meth-
anogenic microorganisms, which survived the acidic pretreat-
ment of the inoculum, started growing and working.
Regarding the VFA profile at 15% w/w, butyric acid was
predominant, followed by acetic acid, around 70 and 25% of the
total VFAs, respectively, after 3 and 5 days. This profile can be
explained considering the chemical composition of OPs, rich in
soluble simple sugars (glucose, sucrose, and fructose), which, as
previously commented, n-hexane was not able to extract along
the previous solid/liquid separation stage, remaining available
for the dark fermentation. A previous research demonstrated
that substrates rich in sugars, such as OPs, at a TS concentration
of around 15−20% w/w, comported the butyric acid production
in a short duration, followed by acetic acid. This latter started to
be predominant for a longer time. This trend was also followed
in our tests, where acetic acid and propionic acid concentrations
increased at the expense of butyric acid32 with prolonging
duration of the fermentation. This presented a further
confirmation of the transition from acidogenic stage to
acetogenic and methanogenic stages, where the produced
VFAs are degrading in acetate and then in methane.33
The results were very different when the TS concentration
was increased to 20% w/w. As commented, the VFA yields were
lower than those observed at 15% TS w/w, with a predominance
of acetic acid as the main component because of the short
duration of the fermentation test. These results were consistent
with the results achieved by Jiang et al.32 and Jankowska et al.,34
who have already observed the reactor instability at a very high
organic matter rate. Moreover, it was fundamental to remark
that the dark fermentation of OPs was characterized by an
inadequate movement of the reaction medium, as a consequence
of the adsorption of the aqueous phase by the increased amount
of OPs. In this condition, water was entrapped in the internal
pores of the OPs, causing the increase of the apparent viscosity
of the reaction medium and inadequate mass and heat transfers,
which reduced the performances of the process.11,19 The system
started to show improved performances after 7 days of
fermentation, when the hydrolysis and the conversion of the
OPs into VFAs finally occurred. In fact, the YVFAs increased to
higher than 25% and the VFA profile was similar to that from
dark fermentation after 3 and 5 days at the TS concentration of
15% w/w.
Finally, the TS increasing at a concentration of 25% w/w
completely inhibited the system, for both short and long
durations. Basically, no VFAs were produced.
Considering these results, the optimal TS concentration and
duration for the VFA production from OPs were 15% TS w/w
and 5 days, respectively. However, the system started showing
inhibition at 20% TS w/w, even if it demonstrated to be able to
react with the high organic matter at longer times (7 days). On
the contrary, at 25% TS w/w, the microorganisms did not seem
to be adapted to support the VFA production.
Biogas Production from OPs. BMP tests were conducted
on OPs after (extracted OP tests) and before (OP tests)
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ACS Sustainable Chemistry & Engineering
Figure 8. Cumulative methane production of OPs and extracted OPs tests.
limonene recovery by n-hexane extraction. Figure 8 shows the
cumulative methane production of both the tests.
Both the curves are characterized by a similar double-stage
behavior: the first stage characterized by a high methane
production kinetics, followed by a first deceleration, and then, a
new increase in the kinetics. This trend can be probably
explained considering the heterogeneous chemical composition
of the OPs, rich in soluble sugars (glucose, fructose), which are
promptly and easily converted into biogas by methanogens. On
the other hand, hemicellulose and cellulose, the other two major
compounds of the OPs accounting for 9−12% w/w and 30−
38% w/w, respectively,28 are known to be more recalcitrant, and
their hydrolysis was slower.
Another interesting result was that the presence of limonene
in OPs did not influence the final values of the specific methane
production, which were 356 NLCH4/kgTVS and 366 NLCH4/kgTVS
for the OPs without and with limonene extraction, respectively.
This result is in contrast to previous research works conducted
on AD of OPs. It was demonstrated that citrus fruits have a
defense system for protection against microbial invasion that
includes the presence of antimicrobial compounds in their peel
(e.g., D-limonene), so citrus waste can inhibit the AD
process.35−37
Calabrò et al.38 confirmed limonene as a toxic compound for
the microorganisms to be removed before the AD process.
Consequently, they conducted semicontinuous AD tests on OPs
after limonene recovery, with an HTR of about 20 days. The
final methane yield was about 200 LCH4/KgTVS, a value close to
the specific methane yield from our extracted OP tests on the
20th day (Figure 8). Carvalho et al.39 adopted a different
strategy to reduce the inhibiting effect of limonene on AD: they
did not recover it from OPs before the AD but conducted a
codigestion of OP with sewage sludge. In this way, they achieved
an improvement of 70% of methane production compared to the
monofermentation of OPs, with a specific methane yield of 300
LCH4/KgTVS. The toxic effect of limonene was also remarked by
another research work,40 where the effects of the organic load
rate (OLR) and temperature were also considered. The authors,
who evaluated the biogas production in a continuous mode in a
pilot system of 84 L, observed that the highest daily specific
methane yield was achieved at an OLR of 1.0 gTVS/Ld and at an
essential oil concentration of about 50 mg/Ld. A partial
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pubs.acs.org/journal/ascecg Research Article
inhibition of the process was observed at an OLR and essential
oil concentration of 2 gTVS/Ld and 85−90 mg/Ld,
respectively. Instead, the system was irreversibly intoxicated at
an OLR and essential oil concentration of around 2.5 gTVS/Ld
and 115 mg/Ld, respectively. Another interesting fact was also
that thermophilic conditions led to a lower methane production
with a decrease of about 75% compared to mesophilic
conditions also for low OLR and essential oil concentration.
The results of the present work lead to a different conclusion:
limonene was not toxic for the microorganisms involving in the
AD process, having simply an inhibiting effect on the kinetics of
the reactions. The limonene removal from OPs favored a
prompter hydrolysis and conversion in methane both of sugars
and carbohydrate with more recalcitrant cellulose, reaching a
final value around the 30th day of the test. This value is
consistent with the typical HRT of semicontinuous and
continuous AD processes. On the contrary, OPs still including
limonene required more time to express its total methane
potential, 45−50 days. This duration allows us to explain why
previous researches, conducted for a shorter time, indicated a
very low methane potential for OPs with limonene.37−39
Finally, these considerations lead to an important conclusion:
limonene recovery is fundamental in terms OP valorization. It
represents a high economically valuable compound, and, in
terms of AD, its presence comports the elongation of the
process, which makes the process not energetically and
economically convenient.
■
CONCLUSIONS
A sequence of chemical−physical and biological unit operations
were proposed for the implementation of a biorefinery scheme
to valorize OPs, an organic waste rich in limonene, a high-added-
value molecule. The OEO extraction was optimized adopting n-
hexane as a solvent at a temperature of 85 °C and with a solvent/
OPs ratio of 2:1. These conditions allowed us to reach an OEOY
of 1.31% with a limonene content of about 90%, values slightly
higher than those in previous works on this topic in the
literature. Considering the high commercial price of limonene,
its numerous applications, and the high degree recovery of the
solvent (78%), the process can be considered economically
convenient. After limonene recovery, the OPs were fermented
for VFA production, achieving good yields (almost 0.35 g VFA/
gTS) at a TS concentration of 15% w/w and after 5 days of
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ACS Sustainable Chem. Eng. 2020, 8, 6834−6843
ACS Sustainable Chemistry & Engineering
fermentation. As an alternative, the extracted OPs can be used
for methane production by AD, reaching a specific methane
production of 365 LCH4/kgTVS. It was demonstrated the
limonene extraction favors a reduction in the duration of the
process, making it more economically convenient. Considering
the good yield obtained, their higher commercial prices, and
their several applications, VFA production can be considered a
better alternative than the mere biogas production for the
exploitation of the OPs.
■
AUTHOR INFORMATION
Corresponding Author
Federico Battista − Department of Biotechnology, University of
Verona, Verona 37134, Italy; orcid.org/0000-0002-3703-
3557; Phone: +39045802765; Email: federico.battista@
univr.it
Authors
Gianmarco Remelli − Department of Biotechnology, University of
Verona, Verona 37134, Italy
Serena Zanzoni − Centro Piattaforme Tecnologiche, University of
Verona, Verona 37137, Italy
David Bolzonella − Department of Biotechnology, University of
Verona, Verona 37134, Italy
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssuschemeng.0c01735
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank the Centro Piattaforme Tecnologiche of the
University of Verona for the NMR characterization of the orange
essential oil.
■
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