Orange Peel Waste Valorisation through Limonene Extraction

using Bio-based Solvents

Baranse Ozturk†, James Winterburn†, Maria Gonzalez-Miquel†‡*

†School of Chemical Engineering and Analytical Science, Faculty of Science and

Engineering, The University of Manchester, Manchester M13 9PL, United Kingdom

‡Departamento de Ingeniería Química Industrial y del Medioambiente, ETS Ingenieros

Industriales, Universidad Politécnica de Madrid, C/ José Gutiérrez Abascal 2, 28006

Madrid, Spain

1

Abstract

Orange peel waste (OPW) can be an effective feedstock for extraction of natural bioactive

components such as limonene, a high value-added chemical broadly exploited for food,

pharmaceutical, and cosmetic industrial applications. Extraction of limonene from OPW has

been conventionally performed via solvent extraction using hexane, a hazardous

petrochemical solvent currently restricted under international regulations. In this work, we

have conducted a comparative assessment of the performance of a variety of green solvents

for sustainable valorisation of OPW through limonene extraction. In particular, cyclopentyl

methyl ether (CPME), ethyl lactate (EL), isopropyl alcohol (IPA), polyethylene glycol 300

(PEG 300), isopropyl acetate (IAc), dimethyl carbonate (DMC), methyl ethyl ketone (MEK),

2-methyl-tetrahydrofuran (2-MeTHF) and ethyl acetate (EAc) have been evaluated as hexane

replacement for the recovery of limonene from OPW. Initially, a preliminary solvent

screening was carried out using the COnductor-like Screening MOdel for Real Solvents

(COSMO-RS) to estimate the solubility of limonene in the proposed solvents and rank their

theoretical extraction performance. Afterwards, experimental studies were performed to

determine the limonene extraction yields and optimize the operating conditions (temperature,

time and solvent load) for limonene recovery from OPW using the various solvents, as

confirmed by gas chromatography mass spectrometry (GC−MS) analysis. Overall, results

support that CPME and 2-MeTHF bio-based solvents significantly outperform the benchmark

petrochemical solvent hexane by increasing limonene extraction yields up to 80% and 40%

respectively at optimum operating conditions. Moreover, recovery and reuse of these solvents

in consecutive extraction cycles was successfully accomplished, while scanning electron

microscopy analysis (SEM) suggests that solvent effects on biomass structure disruption

could be beneficial for further bioprocessing.

Keywords: orange peel waste; limonene; solvent extraction; green solvents;

COSMO-RS.

2

1. Introduction

Food waste has become a major problem worldwide, as roughly one third of all the food

produced for human consumption is wasted every year. In particular, the global volume of

food waste is estimated at 1.6 Gt , accounting for a carbon footprint of 3.3 Gt of CO 2

equivalent and a direct economic cost of USD 750 billion per year [1]. The citrus industry is a

major contributor to food waste, as over 70 Mt of oranges are produced annually worldwide

[2], with juice processing plants generating peel residues up to 50-60% of the total fruit

weight. However, orange peel waste (OPW) is a chemically complex and highly

biodegradable residue, that contains a wide array of valuable compounds, including

fermentable sugars, carbohydrate polymers, flavonoids, polyphenols and essential oils,

providing a unique opportunity for feedstock valorisation as a renewable source of high

value-added chemicals and energy [3,4].

Essential oils are mixtures of aromatic hydrocarbons found in the vesicles located in the

flavedo of citrus peel, possessing organoleptic, antioxidant and therapeutical properties wich

are exploited in a variety of fine chemical, nutritional, and medical applications [5].

Limonene, a highly lipophiphilic cyclic monoterpene, is the main constituent of citrus

essential oil (around 68-98% w/w) and one of the main components of citrus peel waste (up to

4% w/w) [6]. Due to its antioxidant properties and fragrance character [7], limonene is widely

used as preservative agent in food industry as a Generally Recognized As Safe ("GRAS")

additive in the Code of Federal Regulations [8]. In fact, limonene plays a key role in the

global flavour and fragrance market valued at over USD 18.6 billion, with increasing demand

for industrial applications in nutraceutical, cosmetic and pharmaceutical formulations [9].

Moreover, limonene has been identified as an effective bio-based subsitute to volatile organic

compounds (VOCs) in analytical chemistry, as a building block for manufacturing purposes

and as an extraction solvent for separation processes [10,11]. This is of special interest

considering that the bio-based solvent market is expected to reach USD 13.74 billion by 2024

due to current regulations in relation to limit VOC emissions from chemical industrial

sectors, including adhesives, paints and coatings, pharmaceuticals, domestic and industrial

cleaners, and cosmetics among others [12]. Extraction of limonene from citrus peel is also

essential for bioconversion, as this molecule is strongly toxic for fermentative bacteria due to

its antibacterial properties, acting as a microbial growth inhibitor and hindering biofuel

production processes [13,14]. Furthermore, limonene has also been used as green solvent for

3

biomolecule extraction [15] as well as a platform chemical to produce value added products

through oxidation routes [16]. Hence, extraction of limonene from orange waste is an

attractive biorefinery strategy for recovering valuable molecules while promoting further

biological biomass valorization processes.

The design of sustainable extraction processes from natural products is nowadays receiving

increasing attention to improve resource efficiency and cost-effective supply, while reducing

the use and generation of hazardous substances. Following the principles set by Green

Chemistry [17,18], sustainable extraction of natural products should encompass energy

efficient techniques along the use of alternative solvents and renewable feedstocks, while

ensuring safe and high quality product extracts [19]. Solid-liquid extraction has been

proposed as an effective alternative to hydro-distillation, an ancient technique for extraction

of essential oils that requires high energy consumption and long operational times [20,21].

Conventional solvent extraction for limonene recovery has been carried out using hexane as

extractant due to its low boiling point and highly hydrophobic character [22]; however,

hexane is a toxic, petrochemical solvent which causes significant environmental and health

issues, which is currently restricted by different international regulations, such as REACH

(EC 1907/2006), concerning the Registration, Evaluation, Authorization and restrictions of

Chemicals, or IPPC (96/61/EC) regarding Integrated Pollution Prevention and Control. In this

context, the use of benign alternatives to hazardous volatile organic solvents in the chemical

industry plays an important role, such as the so-called green solvents which are those

preferentially produced from biomass feedstock (i.e. bio-based solvents) and/or

environmentally friendly petrochemical solvents that are considered non-toxic and

biodegradable [23]. Our previous works have proved the possibility to valorize citrus waste

using renewable deep eutectic solvents (DESs) for purification of orange essential oils in

downstream processes [24] and extraction of natural antioxidants from orange peel residues

[25]. However, to the best of our knowledge, a systematic study adressing the feasibility of

using green solvents for limonene extraction from citrus waste has not been performed. Thus,

in this work, we assess the posibility of using alternative, bio-based solvents to directly

extract limonene from orange waste to promote sustainable valorization processes of citrus

by-products. This will lead to identify promissing biorenewable solvents with better

extraction peformance than conventional methods to develop citrus waste biorefinery schemes

with improved overall process performance, lowering energy requirements and solvent

4

volumens while increasing product output and quality. Herein, the initial solvent selection

was accomplished by carefully considering Environmental, Health and Safety (EHS)

parameters, as well as physico-chemical properties of a variety of green solvents [26,27]. As a

result, cyclopentyl methyl ether (CPME), ethyl lactate (EL), isopropyl alcohol (IPA),

Polyethylene glycol 300 (PEG 300), isopropyl acetate (IAc), dimethyl carbonate (DMC),

methyl ethyl ketone (MEK), 2-methyl-tetrahydrofuran (2-MeTHF) and ethyl acetate (EAc)

were selected as potential green solvents to replace hexane in the extraction of limonene from

OPW. Solvents such as EAc, IAc and IPA have been identified as less hazardous organic

solvents in the Pfizer and Sanofi selection guides for pharmaceutical applications [28].

Meanwhile, EL is a bio-based, food grade solvent that has been previously used to extract

phytonutrients and phenolic compounds from fruit and vegetable by-products [29,30]. In

addition, EL, DMC and CPME have been proposed for the extraction of oils from yeast [31].

Specifically, CPME and 2-MeTHF, which have been widely recognize as bio-based

alternatives to ether solvents [28,32], have been proved to be especially effective for hexane

replacement in extraction of carotenoids and microalgal lipids [33,34]. MEK is a non-

halogenated solvent that has been suggested to replace chloroform to recover poly(3-

hydroxybutyrate-co-3-hydroxyvalerate) [(PHBV)] from bacterial cells [35] and for crude oil

dewaxing applications [36]. PEG 300 was previously suggested as an efficient limonene

extractant [37], hence it was included in this study for the purpose of comparision.

As there is still lack of information regarding application of green solvents for citrus waste

valorization, the goal of this work is to provide a comparative assessment of the performance

of alternative solvents for limonene recovery. To do so, a preliminary solvent screening was

performed using the COnductor-like Screening MOdel for Real Solvents (COSMO-RS) [38]

as a quantum-chemical approach to estimate the solubility of limonene in the abovementioned

list of preselected 10 solvents (9 green solvents plus hexane as benchmark). Having

completed the computational screening to rank the theoretical solvent performance,

experimental studies were performed by means of solid-liquid extraction to validate the

predicted trends and further optimize the operating parameters for limonene recovery from

actual OPW using the various solvents. Experimental results were evaluated in terms of

limonene extraction yield and the effect of key variables including extraction temperature,

time and solvent load were evaluated to determine the most favourable conditions for

limonene extraction. Limonene is an unsaturated hydrocarbon prone to degradation under

5

influence of heat and light, hence extraction time and temperature are key operating

conditions to maximize extraction yield whilst avoiding degradation. In addition, solvent

recovery and recycling was succesfully accomplished. Lastly, scanning electron microscope

(SEM) evaluation was performed to illustrate the solvent effects on the biomass structure as

this can affect further bioprocessing steps. Overall, this study offers a comprehensive

evaluation of green solvents for citrus waste valorization through extraction of high-value

limonene for market applications, while facilitating further biomass conversion processes for

sustainable citrus waste biorefinery development.

2. Materials and methods

2.1 Plant material

OPW was provided by a local juice bar (Falafel Express, Oxford Road, Manchester, United

Kingdom). The resulting dry orange peel residue was obtained by initially chopping orange

peels and pre-treating with vacuum oven (Vacutherm, ThermoScientific) at 50°C and 150

mbar for 24 hours to remove moisture. Afterwards, liquid nitrogen was applied to avoid

degradation of the components present in the peel following the procedure described

elsewhere [39]. Treated peel was then powdered using a blender and sieved to obtain a

particle size of 1 millimetre (mm).

2.2 Reagents and solvents

The following solvents used in the extraction process were purchased from Sigma Aldrich:

hexane (purity>97%), ethyl lactate (EL) (purity>98%), isopropyl alcohol (IPA)

(purity>99.7%), cyclopentyl methyl ether (CPME) (purity>99%), dimethyl carbonate (DMC)

(purity≥99%), 2-methyl-tetrahydrofuran (2-MeTHF) (purity > 99%), ethyl acetate (EAc)

(purity>99%); isopropyl acetate (IAc) (purity≥97%) was purchased from Alfa Aesar and both

methyl ethyl ketone (MEK) and polyethylene glycol 300 (PEG 300) were purchased from

VWR International ltd. Limonene standard (purity>98%) was purchased from Fisher

Scientific and methanol (purity=99%) was also purchased from Alfa Aesar. Chemical

structures of solvents and limonene are shown in Figure 1.

6

EAc DMC ELHexane

Figure 1. Chemical structures of solvents and limonene.

2.3 Limonene extraction

Limonene was recovered from dry OPW by solid-liquid extraction using hexane (benchmark

petroleum derived solvent) and green solvents i.e. cyclopentyl methyl ether (CPME), ethyl

lactate (EL), isopropyl alcohol (IPA), Polyethylene glycol 300 (PG 300), isopropyl acetate

(IAc), dimethyl carbonate (DMC), methyl ethyl ketone (MEK), 2-methyl-tetrahydrofuran (2-

MeTHF) and ethyl acetate (EAc), structures shown in Figure 1. To preliminary assess the

feasibility of the solvents for limonene extraction, the following solid-liquid extraction

procedure was performed at base case conditions [6,22]: pre-treated OPW was mixed with

each extraction solvent at 1:10 solid to liquid ratio in 15 ml polypropylene centrifuge tubes

and the mixture was mechanically stirred using a shaking incubator (Labnet VorTemp™

1550) for 120 min at the temperature of 30°C at 900 rpm speed. The mixture was then

centrifuged at the same temperature for 10 minutes (Labnet Spectrafuge™ 6C Compact

Research Centrifuge) at 5000 rpm to separate the supernatant. After achieving complete phase

separation, the solvents were evaporated under vacuum in a rotary evaporator. The residue

was dissolved in methanol and then filtered through 0.45-µm filter to remove any remaining

impurities before chromatographic analysis. All experiments were carried out in triplicate.

With the aim of optimizing the extraction conditions, the effect of important operating

parameters including extraction time, temperature and solid to liquid ratio on limonene

recovery from pre-treated OPW was further assessed following a similar extraction procedure.

Extraction time was recorded at every 30 min up to 180 min to find the point at which

equilibrium is reached and solvent is fully saturated, as beyond this time no more solute will

be recovered and hence longer extraction times will be nonsensical [22,40]. The effect of

increasing the extraction temperature from 30 °C up to 50 °C, 70 °C and 90°C on the

limonene recovery yield was subsequently evaluated. Increasing temperature can enhance

solubility of a solute in a chosen solvent which increases extraction yield; however, a

temperature limit of 90°C was set as previous studies [41] have shown that degradation of the

product occurs at temperatures above this point. Furthermore, solid to liquid ratios of 1:5,

1:10 and 1:20 were studied to evaluate the effect of solvent load on the extraction

performance, as this is also an important parameter to maximise extraction yield whilst

avoiding wasting solvent and giving both economic and environmental benefits. Following

the extraction, the solvent was recovered by vacuum distillation using rotary evaporator and 7

sample yield recorded. The % yield of limonene was calculated in terms of dry matter (DM)

as described in Equation (1):

Limonene yield (%)=weight of limonene obtained afterextractionweight of OPW (dry matter ,DM )

× 100

(1)

To evaluate the feasibility of solvent recycling, the recovered solvents were used without any

further purification in subsequent extraction cycles to recover limonene from OPW following

the extraction procedure explained above. The overall extraction procedure followed is

depicted in Figure 2.

Figure 2. Scheme for limonene extraction from orange peel waste (OPW) using bio-based

solvents.

8

2.4 Analytical method for limonene identification

Gas chromatography mass spectrometry (GC−MS) analysis was performed in all the extracts

using Agilent Technologies 5973 equipped with Elite-5ms (30 m x 0.25 mm x 0.25 μm)

column following a method proposed by Bourgou et al [42]. Helium was used as a carrier gas

at a flow rate of 1.2 millilitre per minute (ml/min) and a split ratio of 1:60 using the following

temperature program: rising from 50°C to 240°C at a rate of 5°C/min. The injector and

detector temperature were both maintained at 240°C. Mass spectra were obtained by means of

electron ionization at 70 eV in the range of m/z 40-300 with 1 second mass range. Limonene

was identified as the major and most significant product in all the extracts using mass spectra

in NIST mass spectroscopy library, comparing retention times and mass spectra against the

standard component.

2.5 Scanning electron microscopy

A ZEISS EVO 60 model microscope was used to obtain scanning electron microscope (SEM)

images to visually observe changes occurring at the surface of the samples before and after

the extraction. Samples of dry OPW before and after the extraction were dried overnight to

eliminate any water/moisture, as this may adversely affect microscopic analysis, and later

coated with a thin layer of carbon to improve the sample conductivity. 1 nanometre (nm)

resolution power and 5 kilovolt (kV) acceleration voltage was used to obtain final images.

2.6 Computational Methodology

COnductor-like Screening MOdel for Real Solvents (COSMO-RS) is a quantum-chemical

method to estimate thermodynamic properties and phase equilibrium of pure fluids and its

mixtures based on the prediction of the chemical potential of the compounds [38]. This

approach combines quantum chemical considerations (COSMO) with statistical

thermodynamics (RS) for prediction of thermophysical properties without the need of

experimental data, as calculations are based on information relative to the chemical structure

of the compounds [43]. Hence, this tool results particularly useful to perform preliminary

screenings to evaluate the theoretical performance of novel solvents in separation processes

based on the solubility of target compounds in the liquid phase. In this study, the calculation

9

of the solubility of limonene (x j) in the proposed solvents have been performed using

COSMO-RS model as per Equation (2) [44]:

log10 ¿) = log10[exp ((µ jpure−µ j

solvent−ΔG j ,fusion ) /RT ) ] (2)

where µ jpure is the chemical potential of pure compound j, µj

solvent is the chemical potential of j

at infinite dilution and ΔG j , fusion is the free energy of fusion of j.

All COSMO-RS calculations were performed using COSMOthermX software, version C30,

release 1601 at the BVP86/TZVP/DGA1 quantum chemical level and (BP_TZVP_C30_1601)

parametrization as described elsewhere [45–47]. Previous works have also used COSMO-RS

to predict the solubility values of carotenoids and lipids in green solvents [31,33,48] as well

as to model the thermodynamic behaviour of terpenic hydrocarbon mixtures [24].

3. Results and Discussion

3.1 Preliminary assessment of solvent performance: COSMO-RS vs Experimental Evaluation

COSMO-RS simulations were conducted to perform an initial assessment of the capability of

the proposed green solvents to replace the petrochemically derived hexane to extract limonene

from OPW, as presented in Table 1.

Table 1. COSMO-RS relative solubility, log10(xi), and probability of solubility of limonene, P, in various

solventsa

Calculations have been performed in terms of the relative solubility, log(x i), of limonene in

the various solvents, setting to 0 the logarithm of the solvent with the highest solubility value

so all other solvents are given values relative to this; afterwards, this algorithm is converted

10

Solvent log10(xi) P (%)Hexane -0.0657 86Cyclopentyl methyl ether (CPME) 0.0000 100Ethyl lactate (EL) -0.2881 52Isopropyl alcohol (IPA) -0.5730 27Isopropyl acetate (IAc) -0.1048 79Dimethyl carbonate (DMC) -0.4480 36Methyl ethyl ketone (MEK) -0.1869 652-methyltetrahydrofuran (2-MeTHF) -0.0029 99Ethyl acetate (EAc) -0.1687 68Polyethylene glycol (PEG) -0.0376 92

Higher than reference Lower than referenceReferencea

into probabilities (%) [33]. Solvents that present log (xi) values close to 0 are the most feasible

solvents, hence have higher probability values. Therefore, these results provide a relative

solubility ranking that represent trends of the solubility of the target solute, i.e. limonene, in

the proposed solvents. As presented in Table 1, the probability of solubility of limonene in the

reference solvent hexane is 86%, while there are three solvents with higher probability values,

which are CPME (P=100%), 2-MeTHF (P=99%) and PEG (P=92%). Therefore, considering

the global computational results, CPME followed by 2-MeTHF appear to be promising bio-

based solvents to replace hexane for limonene extraction from citrus peel waste among the

proposed green solvent systems. Subsequently, all proposed solvents were experimentally

employed to perform the actual extraction of limonene from citrus peel waste to validate the

computational trends predicted by COSMO-RS.

Hex

ane

CPM

E EL IPA

PEG

300 IA

c

DM

C

MEK

2-M

eTH

F

EAc

0.0

0.5

1.0

1.5

2.0

Base case (t=120 min, T=30 °C, S/L=1:10)

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100)

Figure 3. Limonene yield (%) from OPW (dry matter, DM) at base case experimental operating conditions (t= 120 min, T = 30°C, S/L = 1:10) using various extraction solvents.

The experimental results, which are graphically represented in Figure 3, support that all the

proposed solvent systems are capable of extracting limonene from OPW; however, solvent

performance to replace hexane showed variations. The limonene extraction performance

under benchmark conditions (t= 120 min, T = 30°C, S/L = 1:10) using the various extraction

solvents is as follows: CPME > 2-MeTHF > PEG 300 > IAc > Hexane > EAc > MEK > EL >

DMC > IPA. In particular, CPME has significantly outperformed the benchmark solvent 11

(hexane) by providing 0.81% limonene yield, this is 1.5 fold increase in comparison with the

amount extracted by hexane (0.53% limonene yield). CPME is an aprotic dipolar solvent

[26], which means that promotes the dissolution of any compound with an alkane chemical

structure such as limonene; in fact, previous studies have shown that CPME performs

particularly well for the extraction of carotenoids, components that exhibit alkane-based

structure [33]. Additionally, 2-MeTHF yielded 0.64% of limonene, which represents 1.2 fold

increase in comparison with the solute recovery yield provided by the benchmark solvent

hexane. 2-MeTHF is also an aprotic dipolar solvent which has been effective in extracting

vegetable oils from food crops [49] and lipids from microalgae [34] due to the favourable

solvent extraction kinetics, i.e. starting accessibility and effective diffusivity, enabling

solvation of higher amount of solute at the surface with faster extraction rate than hexane

[33]. Note that aprotic highly dipolar solvents like DMC, with moderate hydrogen bonding

strength, or amphiprotic solvents including EL or IPA, with higher hydrogen-bond

donor/acceptor ability, have a lower performance than hexane for limonene extraction; this is

due to the increased polarity of those solvents, which are not capable of favourably interacting

with the non-polar limonene solute; therefore, upon mixing, terpenic hydrocarbon molecules

will stay associated through London dispersion forces and neat solvent molecules will interact

through hydrogen bonding, hence limiting effective solute-solvent molecular interactions,

resulting in low extraction yields.

Overall, considering the theoretical limonene solubility trends computed using COSMO-RS

method and the experimental data obtained for actual extraction of limonene from OPW, it

can be concluded that COSMO-RS predictions were consistent with the experimental

observations, thus validating this computational tool for screening of novel solvents for

limonene extraction.

3.2. Optimisation of limonene extraction operating conditions

3.2.1. Extraction time

The change in limonene extraction yield was evaluated as a function of the extraction time

starting from 30 min up to 180 min keeping other operating conditions at base case (T=30°C

and S/L=1:10). Results in Figure 4 show that the limonene extraction yields progressively

increase up to 150 min, and afterwards the change in the extraction yield is significantly less

noticeable. This could be explained in terms of the Fick’s second law of diffusion, as mass

12

transfer of the solute from biomass to solvent phase only occurs until the system reaches

equilibrium [50]. Additionally, due to the nature of limonene, prolonged extraction times

could lead to degradation of this unsaturated compound with the influence of light, heat and

air. In this study, from 30 min to 150 min, the limonene yield using hexane increased from

0.31% to 0.65%, which represents over two-fold extracted limonene; and with the best two

solvents, the limonene extraction yield has changed from 0.63% to 1.14% when using CPME

and from 0.49% to 0.84% when using 2-MeTHF, representing increases of 81% and 71%

respectively. Meanwhile, between 150 and 180 min, the limonene recovery only increased by

1.7% with hexane, 2.5% with CPME and 6% with 2-MeTHF. Therefore, due to the minimal

increase in limonene yield from 150 to 180 min and to avoid degradation of product, 150 min

was chosen as the optimal extraction time.

It is worth noting that stirring rate was set to a high value in all the experiments to favour

contact between biomass and solvent phases in order to enhance mass transfer and reducing

extraction times. However, if lower stirring rates are used, longer extraction times may be

required, which could promote product degradation.

Hex

ane

CPM

E EL IPA

PEG

300 IA

c

DM

C

MEK

2-M

eTH

F

EAc

0.0

0.5

1.0

1.5

2.0

30 min 60 min 90 min 120 min 150 min 180 min

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100)

Figure 4. Limonene yield (%) from OPW (dry matter, DM) as a function of the extraction time (at T = 30°C and

S/L = 1:10) using various extraction solvents.

3.2.2 Extraction temperature13

Extraction of limonene from OPW with proposed solvents via solid-liquid extraction was

examined under the temperatures of 30, 50, 70 and 90 °C, by keeping other operating

conditions at base case (t=120 min and S/L= 1:10), to understand the influence of heat on the

extraction ability of the solvents but also to find a limit to avoid degradation of the target

limonene product. Generally, as we can see from the results in Figure 5, increasing the

operating temperature up to 70°C boosted the extraction efficiency of the process, which

could be due to the increased solubility and diffusion coefficients of the target compound in

the solvents [51] along the reduced surface tension and viscosity of the solvent systems,

promoting mass transfer of the solute from solid to liquid phase. Furthermore, at a higher

temperature, speed of the molecular movements is faster, which causes the extracting agent to

diffuse more quickly into the sample and similarly, solute to diffuse faster from biomass into

the solvent. However, at 90°C, limonene degradation starts occurring as evidenced from the

product extraction yield decreasing gradually. Previous researchers [41] suggested that 70°C

is the highest appropriate temperature for terpene extraction to avoid degradation of products.

Results have revealed that with hexane, limonene extraction yield was about 0.99% at 70 °C

but decreased to 0.91% limonene yield at 90 °C. Additionally, the limonene extraction yield

provided by CPME was 1.69% at 70 °C, and reduced to 1.51% at 90 °C. Likewise, the

limonene recovery yield provided by 2-MeTHF was reduced from 1.34% at 70 °C to 1.28%

90 °C. According to previous studies, 25% terpene degradation occurs at 30 min of heating

time at 100°C [52], and 50% terpene degradation occurs at 24 h of heating time at 120°C [53].

Therefore, an extraction temperature of 70°C is deemed appropriate to enhance the limonene

recovery yield while minimizing product degradation.

14

Hex

ane

CPM

E EL IPA

PEG

300 IA

c

DM

C

MEK

2-M

eTH

F

EAc

0.0

0.5

1.0

1.5

2.0

30°C 50°C 70°C 90°C

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100)

Figure 5. Limonene yield (%) from OPW (dry matter, DM) as a function of the extraction temperature (at t=120

min and S/L = 1:10) using various extraction solvents.

2.3. Solid/liquid ratio

Operating and capital costs are significantly affected by the solvent load used in the

extraction plant, hence solid/liquid (S/L) ratio is an important parameter to consider for

process profitability. Different S/L ratios of 1:5, 1:10 and 1:20 were studied whilst keeping

other conditions at base case (t=120 min, T=30°C) to evaluate the amount of solvent required

to enhance the limonene recovery yield without compromising the cost factor. As seen in

Figure 6, the limonene yield with all solvents progressively increases with the amount of

solvent used, as this promotes contact between both phases, which enhance extraction until

the equilibrium is reached. Moreover, higher S/L ratios increase the concentration gradient

and diffusion rates promoting the extraction ability of the solvent.

Although using a S/L ratio of 1:20 provides the highest extraction yield, operating with large

quantities of solvent will increase energy requirements for heating and pumping, material and

equipment cost, as well as effluent disposal while providing less concentrated extracts, which

may limit the profitability of the plant. Increasing the S/L ratio from 1:5 to 1:10 enhanced the

limonene extraction yield from 0.29% to 0.53% for hexane, from 0.66% to 0.81% for CPME,

and from 0.49% to 0.64% for 2-MeTHF. Moreover, when using S/L ratios of 1:10, CPME and 15

2-MeTHF outperformed hexane by providing higher limonene recovery. Therefore, 1:10 was

chosen as a suitable ratio to enhance the efficiency of the limonene extraction process for the

reasons explained above.

Hex

ane

CPM

E EL IPA

PEG

300 IA

c

DM

C

MEK

2-M

eTH

F

EAc

0.0

0.5

1.0

1.5

2.0

1:5 1:10 1:20

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100

)

Figure 6. Limonene yield (%) from OPW (dry matter, DM) as a function of the solid/liquid ratio (at T = 30°C

and t=120 min) using various extraction solvents.

3.2.4. Optimal extraction conditions

Selecting appropriate operating conditions is vital to enhance limonene recovery from OPW

while reducing time, costs and environmental impact of the extraction process. In this study,

extraction time, temperature and solid/liquid ratio have been evaluated to optimize the

operating parameters for orange waste valorisation through limonene recovery, with results

supporting 150 min of extraction at 70 °C and using solid/liquid ratio of 1:10 as favourable

conditions for such purpose. Accordingly, limonene extraction essays were conducted for all

solvents under the aforementioned optimal operating conditions, as presented in Figure 7.

Based on these results, it is possible to conclude that both bio-based solvents CPME and 2-

MeTHF significantly outperform hexane by providing limonene extraction yields of 1.78%

and 1.37% respectively, while hexane yielded 0.99% of limonene. This means that CPM

16

increased the limonene recovery yield nearly 80% whereas 2-MeTHF increased the limonene

recovery to around 40% in comparison to the benchmark organic solvent hexane.

Additionally, PEG 300 was able to slightly enhance the limonene recovery, yielding 1.17%;

while IAc, MEK and EAc provided limonene yields of 0.99%, 0.94% and 0.98% respectively,

which are similar to those obtained with hexane. Remaining solvents, i.e. EL, DMC and IPA,

were not able to reach the limonene extraction yields provided by hexane as a reference

solvent. Furthermore, when comparing limonene extraction results at optimal conditions

(Figure 7) to base case conditions (Figure 3), it can be noted that by setting more favourable

extraction parameters the limonene recovery efficiency was significantly enhanced, increasing

over two-fold for the best solvent candidates, i.e. CPME and 2-MeTHF.

Hex

ane

CPM

E EL IPA

PEG

300 IA

c

DM

C

MEK

2-M

eTH

F

EAc

0.0

0.5

1.0

1.5

2.0

Optimum (t=150 min, T=70 °C, S/L=1:10)

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100)

Figure 7. Limonene yield (%) from OPW (dry matter, DM) at optimum experimental conditions (t= 150 min, T

= 70°C, S/L=1:10) using various extraction solvents.

When comparing these results to previous works, it can be confirmed that the limonene

extraction yield provided by hexane in this work (0.99 % w/w) is similar to the limonene

extraction yield reported for hexane at similar operating conditions (0.91% w/w) [54].

Furthermore, bio-based solvents proposed in this work provide comparable limonene

recovery from orange peels than conventional techniques such as hydrodistillation (nearly 2%

w/w) [6]. 17

3.3. Biomass surface characterization

The cell wall disruption during the extraction process was explored through Scanning

Electron Microscopy (SEM) analysis on dried OPW biomass before and after the extraction,

employing hexane as benchmark solvent along the best two green solvents (CPME and 2-

MeTHF) under optimal extraction conditions (t=150 min, T=70°C and S/L=1:10). As shown

in Figure 8, biomass cells before the extraction present uniform and well-defined shapes with

large particles due to the presence of complex linkage formed of long cellulose,

hemicellulose, pectin and lignin polymer chains. Meanwhile, biomass treated with hexane

shows smaller particles with less defined shapes due to the chemical disruption after the

biomass contacted with the extracting solvent. These modifications in cell wall structure, such

as shrinking and wrinkling with smaller particles, are more noticeable for CPME and 2-

MeTHF due to their greater ability to break the complex linkages and enhance the limonene

extraction yields. This is in agreement with previous studies suggesting that extraction

conditions and solvent nature can greatly affect biomass disruption and recovery of target

compounds [34,40,55]. In fact, CPME and 2-MeTHF, bio-based solvents that provided higher

limonene yield than hexane, also display more efficient chemical disruption which could be

beneficial for further biomass processing towards promoting integrated bio-refineries for

citrus waste valorisation.

18

DC

BA

Figure 8. SEM images of dry OPW biomass (A) before solvent extraction and after the solvent extraction with

(B) hexane, (C) CPME and 2-MeTHF (D) under optimal extraction conditions (t=150 min, T=70°C and

S/L=1:10).

3.4. Solvent recyclability

Recyclability of the extraction solvent plays an important role in developing sustainable

biomass valorisation processes considering economic and environmental factors. To evaluate

the possibility to recover the solvent after the extraction and reuse it within the process, three

consecutive extraction cycles were carried out under optimal conditions using hexane as

benchmark solvent along the best two green solvents, CPME and 2-MeTHF, for comparison

purposes. As seen in Figure 9, the decrease in the limonene extraction yield from cycle 1 to

cycle 2 was about 42% when using hexane, whereas such reduction was around 20% for

CPME and 25% for 2-MeTHF. In addition, when going from cycle 2 to cycle 3, the

magnitude of the decrease in the limonene extraction yield grows to 55%, 32% and 40% for

hexane, CPME and 2-MeTHF respectively. These results confirm that the recyclability of the

proposed solvents, CPME and 2-MeTHF, is more efficient than that of hexane; in particular,

the limonene extraction yields provided by CPME and 2-MeTHF in the third extraction cycle

were 0.97% and 0.59% respectively, in comparison to 0.25% obtained with hexane. Overall,

this further supports the suitability of replacing hexane with the proposed bio-based solvents

to develop sustainable processes for citrus waste valorisation.

19

Hex

ane

CPM

E

2-M

eTH

F

0.0

0.5

1.0

1.5

2.0

Cycle 1 Cycle 2 Cycle 3

Lim

onen

e yi

eld

(%)

(g li

mon

ene

/ g D

M *

100

)

Figure 9. Limonene yield (%) from OPW (dry matter, DM) using hexane, CPME and 2-MeTHF in three

consecutive extraction cycles.

20

Conclusions

This study demonstrates that green solvents such as bio-based CPME and 2-MeTHF are

promising alternatives for replacing hexane in the extraction of limonene from orange peel

residues to develop sustainable bio-refineries for citrus waste valorisation. At optimum

extraction conditions (T = 70°C, t=150 min, S/L=1:10), CPME and 2-MeTHF respectively

increased limonene extraction yields up to 80% and 40% in comparison with the benchmark

organic solvent hexane. Moreover, recyclability of these solvents within the process was

successfully accomplished, with CPME and 2-MeTHF providing 4-fold and 2-fold limonene

extraction yields in comparison to that provided by hexane after three consecutive extraction

cycles. Additional microscopy analysis suggests that both solvents promote biomass structure

disruption, which could be beneficial for further bioprocessing. Furthermore, COSMO-RS

method was proved an effective tool to conduct preliminary solvent screenings and rank the

theoretical performance of green solvents for limonene extraction.

ASSOCIATED CONTENT

Supporting Information

Figures S1 and S2: The mass spectra showing peaks of limonene standard (A), limonene

extracted with hexane (B), CPME (C) and 2-MeTHF (D).

Acknowledgement

Baranse Ozturk would like to thank EPSRC (Engineering and Physical Sciences Research

Council) for the Ph.D. scholarship provided.

21

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