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Reduction of phorbol esters in Jatropha curcas L. pressedmeal by surfactant solutions extraction

Naphatsarnan Phasukarratchai a,b, Veerapat Tontayakom c, Chantra Tongcumpou a,d,*aCenter of Excellence on Hazardous Substance Management, Chulalongkorn University, Pathumwon, Bangkok 10330, Thailandb International Postgraduate Programs in Environmental Management, Graduate School, Chulalongkorn University, Pathumwon,

Bangkok 10330, ThailandcDepartment of Innovation and Technology, PTT Chemical Public Company Limited, Rayong, ThailanddEnvironmental Research Institute, Chulalongkorn University, Pathumwon, Bangkok 10330, Thailand

a r t i c l e i n f o

Article history:

Received 1 November 2011

Received in revised form

7 May 2012

Accepted 14 May 2012

Available online 21 June 2012

Keywords:

Jatropha curcas L.

Phorbol esters

Surfactant

HLB

Toxin removal

* Corresponding author. Environmental Rese2188138; fax: þ66 2 2188210.

E-mail addresses: tchantra@chula.ac.th, d0961-9534/$ e see front matter ª 2012 Elsevhttp://dx.doi.org/10.1016/j.biombioe.2012.05.0

a b s t r a c t

Phorbol esters (PEs), a compound group found in Jatropha curcas seeds, are toxic and

thermal-resistant compounds; thus, the further application of Jatropha pressed seed is

limited to use only as soil amendment, even though the meal has high protein content,

similarly to that of soy bean meal. This study introduces a technique to remove PEs from

Jatropha meal using a surfactant aqueous-based solution. Both single and mixed surfactant

systems were evaluated. The Hydrophileelipophile Balance (HLB) of a nonionic surfactant

was found to be related to its PEs removal efficiency. The average initial level of PEs in the

mechanically-pressed meals was 1.45 mg g�1. The five systems used to reduce the mass

fraction of PEs were as follows: water; 40 mmol L�1 polyoxyethylene (20) sorbitan mono-

oleate (Tween 80); mixed of 40 mmol L�1 Tween 80, 5 mmol L�1 sodium bis (ethylhexyl)

sulfosuccinate (AOT), and 100 mmol L�1 NaCl; 40 mmol L�1 fatty alcohol C12e14 extended

with 9 ethoxylates (Dehydol LS9); and mixed of 40 mmol L�1 Dehydol LS9, 5 mmol L�1 AOT,

and 100 mmol L�1 NaCl; these systems removed 22.49%, 81.43%, 81.23%, 81.87%, and 78.85%

from the initial meal, respectively. The optimal extraction time was 15 min, and the

removal of PEs was enhanced by the application of a double extraction procedure. This

technique is promising because mass fraction of the PEs was reduced from the initial meal

almost 90%; this approaches the level of PEs found almost as low as those found in a non-

toxic variety of Jatropha seed.

ª 2012 Elsevier Ltd. All rights reserved.

1. Introduction seed can be used in automobile engines directly or indirectly.

Jatropha curcas is a plant in the Euphorbiaceae family and is

native to countries in Central and South America [1,2]. The

morphological characteristics of it’s stems, leaves, flowers,

fruits, and seeds can vary greatly, yet it’s seeds generally

contain 30e40% oil by weight. The oil obtained from J. curcas

arch Institute, Chulalongk

rchantra@gmail.com (C.ier Ltd. All rights reserve20

In addition, the seed meal is nutrient-rich. The mass fraction

of crude protein contained in J. curcas kernels, approximately

26.0% [3], is higher than the percentage found in soybeans [4].

After the oil is extracted from the J. curcas kernels, most of the

protein still exists in the residual seed meal, which can be

used further. Unfortunately, most varieties of J. curcas seeds

orn University, Pathumwon, Bangkok 10330, Thailand. Tel.: þ66 2

Tongcumpou).d.

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 6 49

contain some toxic compounds. For example, phorbol esters

(PEs) can be found in J. curcas seeds and kernels [2,5]. Phorbol

esters are derivatives of the tigliane compound; the primary

structure is a tetracyclic diterpene as shown in Fig. 1(a).

J. curcas seeds contain several different compounds that

belong to the PEs group; these compounds include

derivatives of 12-deoxy-16-hydroxyphorbol (Fig. 1(c)), 12-

deoxy-16-hydroxyphorbol-13-acylate [6], and 12-deoxy-

16-hydroxyphorbol-40-[120,140-butadienyl]-60-[160,180,200-non-atrienyl]-bi-cyclo[3.1.0] hexane-(13-o)-20-[carboxylate]-(16-o)-30-[80-butenoic-100]ate (DHPB, Fig. 1(d)) [7]. However, 12-o-tet-

radecanoyl-phorbol-13-acetate (TPA) is generally used as the

external standard for determining the concentration of

phorbol esters by HPLC [3,8]. The molecular weight of TPA is

616.92 g mol�1, and its chemical formula is C36H56O8. The

structure of TPA is shown in Fig. 1(b).

PEs are considered to be toxic and are suspected carcino-

gens [9]. They are easily absorbed into the body by ingestion

and the dermal route. The possible effects of contact with PEs

include the severe irritation of tissues (e.g., the skin, eyes,

mucous membrane, and lungs) and induced sensitivity [9].

Consequently, the oil extracted from the J. curcas seeds and

Fig. 1 e The structure of PEs: (a) general structure, (b) 12-o-Tetr

hydroxyphorbol, and (d)12-deoxy-16-hydroxyphorbol-40-[120,14hexane-(13-o)-20-[carboxylate]-(16-0)-30-[80-butenoic-100]ate (DHP

the residual meal obtained after the extraction cannot be

safely ingested unless the toxin has been removed.

In order to optimize the use of J. curcas seeds, the PEs

should be removed from both extracted oil and residual meal.

The detoxification of the J. curcas residual meal by a heat-only

treatment may not be appropriate for PEs because they are

thermoresistant. While organic solvent and chemical treat-

ments have the ability to reduce PEs in the meal [10,11]. Are-

gherore et al. [10] found that up to 95% of PEs in residual meal

can be removed by combining a heat treatment with four

washes with 92% methanol by volume; however, the authors

revealed that this technique was not economically feasible.

According to Nokkaew et al. [11], the technique was performed

by the meal washing with 2e3% by weight of potassium

hydroxide for 45 min at room temperature; then followed by

an incubation 95% by volume of ethanol overnight can

removes PEs from J. curcas meal to a lower level as found in

a non-toxic variety of J. curcas. For the oil, solvent extraction is

an effective PEs removal method; according to Devappa et al.

[12,13], a four-time washing with methanol can remove 95%

by mass fraction PEs from J. curcas oil and this treated oil has

no adverse effect to snail.

adecanoyl-phorbol-13-acetate (TPA), (c) 12-deoxy-16-0-butadienyl]-60-[160,180,200-nonatrienyl]-bi-cyclo [3.1.0]

B).

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 650

Although PEs are toxic compounds, they are used in some

applications such as an ingredient in pesticides and insecti-

cides. Rug and Ruppel [14] compared the toxic activities of the

three types of liquid obtained from J. curcas; they observed the

methanol extract, the aqueous extract and the J. curcas crude

oil on intermediate snail hosts and the larvae of schistosomes.

They found that the methanol extract was the most toxic to

the snails at LC50 ¼ 5 mg L�1 and LC100 ¼ 25 mg L�1, while

Bayluscide�, a commercial pesticide for killing snails, can kill

all snails at 1 mg L�1. PEs is one of the toxins in J. curcas that

likely mimics the toxic activity of these pesticides [14].

Naksuk et al. [15] proposed a new approach for the

extraction of palm kernel seeds using a surfactant solution;

they found that the yield of the extracted oil and its quality

were relatively compatible to that obtained after hexane

extraction. This surfactant approach is based on the

microemulsion phenomenon in which the oil is detached

from palm kernel seeds by lowering the interfacial tension

[15]. A similar approach was introduced in this study in

order to separate PEs from J. curcas meal instead of using

volatile solvent and chemical agent. This technique is

possible because a surfactant naturally has a hydrophilic

head and hydrophobic tail, which makes its molecules

soluble in both aqueous and hydrophobic oil environments.

This allows the surfactant to reduce the interfacial tension

between the two phases. In addition, once micelles are

formed, hydrophobic compounds can be trapped within the

micelles [16e18]. As PEs are likely hydrophobic compounds

and less miscible with water [10]; a surfactant can be used

to free the compounds by reducing the interfacial tension

between the compounds and the substrate meal, subse-

quently detaching the PEs from the meal. The objectives of

the present work are to investigate systems and conditions

of surfactant solutions for the removal of phorbol esters

from the J. curcas residual meal. In addition, the effect of

this approach on reduction of crude proteins content in the

meal was also evaluated.

Fig. 2 e The structure of surfactants in this study.

2. Materials and methods

2.1. Materials

2.1.1. J. curcas seed mealThe J. curcas seed meal used in this study was supplied by PTT

Chemical Public Company Limited, Thailand (PTTCH). The

seeds were collected from the PTT Chemical PCL demonstra-

tion plot located at Ta Sit, Pluak Daeng, Rayong, Thailand

(geographical coordinates: 13�0101600 N, 101�1500300 E). It was

collected in August 2007 from mature J. curcas trees (approx.

age 3 years) for the experiment on evaluation of the PEs

removal efficiency by different surfactant solutions and in

January 2009 (approx. age 4.5 years) for the experiment on

evaluation of the effect of salt and physical parameters. The

seed was twice defatted by a PTTCH screw-pressing machine

to make the pressed meal a day after it was collected and

stored in dark seal bags andwas kept cool until it was used for

the experiment. The laboratory experiments were conducted

from April 2008 to February 2009 at Chulalongkorn University,

Bangkok, Thailand.

2.1.2. SurfactantsTwo types of surfactants, nonionic and anionic surfactants,

were used in this study. The nonionic surfactants used were

a commercial grade Dehydol group or fatty alcohol C12e14

extended with 3, 7 and 9 ethoxylates (LS3, LS7 and LS9 with

their CAS Numbers: 68439-50-9,68539-50-9 and 68439-50-9,

respectively). They were supplied by PTTCH Co. LTD. Other

nonionic surfactant used in this study was polyoxyethylene

(20) sorbitan or Tween group such as, polyoxyethylene (20)

sorbitan monolaurate or Tween 20, CAS Number: 9005-64-5

(T20) and polyoxyethylene (20) sorbitan monooleate or

Tween 80, CAS Number: 9005-65-6 (T80) were purchased

from Ajax Finechem; and polyoxyethylene (20) sorbitan

monopalmitate or Tween 40, CAS Number: 9005-66-7 (T40);

and polyoxyethylene (20) sorbitan monostearate or Tween

60, CAS Number: 9005-67-8 (T60) were purchased from

Merck. The anionic surfactant was sodium bis (ethylhexyl)

sulfosuccinate, CAS Number: 577-11-7 (Aerosol OT, AOT)

was purchased from Fluka. The structures of surfactants

are illustrated in Fig. 2.

2.1.3. ElectrolyteThe sodium chloride (NaCl) used was of analytical grade and

obtained from UNIVAR.

2.1.4. SolventsAcetonitrile-grade HPLC was used for the analysis of the

phorbol esters.

2.1.5. Standard phorbol esterThe phorbol ester standard was 12-o-tetradecanol-phobol-13-

acetate, or TPA, CAS Number: 16561-29-8; it was of 98% purity

and was purchased from Fluka.

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 6 51

2.2. Methods

2.2.1. Extraction of PEs from the meal by surfactant solutionsIn order to identify the suitable surfactant systems for PEs

removal, nonionic surfactant systems (0e100 mmol L�1) and

mixed of nonionic (40 mmol L�1) and anionic (0e40 mmol L�1)

surfactant systems with varying concentrations of nonionic

and anionic surfactants were studied. For each system, 2 g of

ground meal were mixed with 20 mL of the surfactant solu-

tion; the mixture was shaken for 30 min at room temperature

(around 30 �C). Next, the residual meal was filtered. The

remaining meal was collected, and the PEs content was

determined by HPLC-UV.

2.2.2. Determination of the PEs content in the mealThe PEs content in the meal before and after extraction with

each surfactant solution was determined. Two-gram samples

were extracted with 20 mL of methanol using an GFL orbital

shaker Model 3017 at 31.42 rad s�1 for 4 h. This extraction

method was verified and found to recover 88.1% of PEs [19].

The extracted samples were analyzed by HPLC-UV (Shi-

madzu-10A VP) following the reverse column procedure

described byHass andMattelbach [8]. Octadecyl (C18) was used

as the functional group, the temperature of column was

controlled at 25 �C, and an 80:20 (v/v) isocratic acetonitrile-to-

water ratio was used as the mobile phase at a flow rate of

1 mL/min. The detector used was a UV adsorption detector at

a wavelength of 280 nm. The injected sample volume was

20 mL. The calibration curve was prepared by dissolving 12-o-

tetradecanoyl-phorbol-13-acetate (TPA), the external stan-

dard, in methanol.

The PEs reduction efficiency in this study is calculated from

the reduction of mass fraction of PEs after extraction by

a surfactant solution at various conditions from the initial

mass fraction of PEs in pressed meal.

2.2.3. Statistical analysisAll experiments were carried out in triplicate. Standard devi-

ation for each set of experiment was calculated and show in

graphic results. The statistical analysis was based on one-way

analysis of variance (ANOVA) for the comparison statistically

significances using the SPSS (Statistical Product and Service

Solutions) version 17.0 software package to compare between

different conditions at *P < 0.05.

Table 1 e Properties of the nonionic surfactants and theirmass fraction reduction of phorbol esters (PEs) (%) using40 mmol LL1 of various types of nonionic surfactants.

Nonionicsurfactant

Properties Mass fraction reductionof PEs (%)

C-chain EON HLB % SD*

Dehydol LS3 C12e14 3 7.9 33.42a 10.11

Dehydol LS7 C12e14 7 12.1 58.01b 2.93

Dehydol LS9 C12e14 9 13.4 65.63bc 4.85

Tween 20 C12 20 16.7 61.41b 4.65

Tween 40 C16 20 15.6 70.87c 2.18

Tween 60 C18 20 14.9 72.14c 1.87

Tween 80 C18 ¼ 1 20 15.0 72.01c 2.31

Note: Superscript a, b and c represent the statistic testing ( p< 0.05)

and * Standard deviation (SD).

3. Results and discussion

3.1. PEs reduction by surfactant solutions

In order to evaluate the efficiency of the removal of PEs from

Jatropha meal, two types of surfactant solutions, a single

nonionic surfactant and a mixed surfactant containing both

nonionic and anionic surfactants, were utilized in this study.

The nonionic surfactants were selected to remove the PEs

because nonionic surfactants generally have lower CMCs than

anionic surfactants, making themmore suitable for removing

oily compounds from the soil or solid surfaces [20,21]. The two

groups of nonionic surfactants selected for this studywere the

Tween group and the Dehydol group. The anionic surfactant

used for mixing with the nonionic surfactant was Aerosol OT

(AOT). The most suitable nonionic surfactant concentration

for the reduction of PEs was 40 mmol L�1; this was derived

from a preliminary investigation. Accordingly, a suitable

nonionic surfactant was selected to bemixedwith the anionic

surfactant and the neutral electrolyte (NaCl). The results of

the evaluation for the nonionic andmixed surfactant systems

are described below.

3.1.1. PEs reduction by single nonionic surfactant systemsThe initial level of PEs in the pressed meal was 1.45 mg g�1 as

TPA. By using the conditions described earlier, the reduction

of PEs mass fraction in the pressed meal ranged from 61% to

72% by the surfactant solutions prepared from the Tween

group and 33e65% by those from the Dehydol group (fatty

alcohol ethoxylate). The individual properties and PEs reduc-

tion efficiency of each nonionic surfactant are shown in

Table 1. The PEs reduction results within most of the Tween

group, including T40, T60 and T80, did not differ significantly

(at a 95% confidence level, p < 0.05). Similarly, the differences

among the PEs reduction results for T20, LS7 and LS9 were

insignificant ( p < 0.05). However, the PEs reduction efficiency

from surfactant solutions of the Tween group T40, T60 and

T80 was found significantly different with the solution of

Dehydol group LS3 and LS7. Among six surfactant solutions of

the two series nonionic surfactant group, LS3 system had the

lowest efficiency.

To better understand the relationship between the

surfactants’ characteristics and their PEs removal perfor-

mances, plots were generated that compared either the

ethoxylate number (EON) of the fatty acid ethoxylate or the

carbon chain length of the tails in the Tween group with the

reduction efficiency; these data are shown in Fig. 3(a). It is

obvious that the EON in a surfactant’s structure plays a larger

role in PEs reduction than the length of its carbon chain. An

increase of each ethoxylate group increased the PEs reduction

efficiency by approximately 5.5%, while an increase of each

carbon chain length in the surfactant’s tail only enhanced the

removal efficiency by approximately 1.8%. The EON repre-

sents the hydrophilic portions of nonionic surfactants; hence,

increasing the EON of a surfactant is expected to enhance the

solubilization of a polar compound. According to Rosen [21],

y = 1.7775x + 40.667

y = 5.4796x + 17.649

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20

PE

s red

uctio

n (%

)

C-Chain length/EON of surfactants

a

C-Chain length EON

0102030405060708090

100

0 5 10 15 20

PE

s red

uctio

n (%

)

HLB of surfactants

b

LS7

LS9

T60, T80T40

T20

LS3

Fig. 3 e Effects of (a) C-chain length and EON, and (b) HLB of

surfactants on the mass fraction reduction of PEs (%) by

40 mmol LL1 of different nonionic surfactant solutions.

50

55

60

65

70

75

80

85

90

95

100

0 2.5 5 7.5 10 20 30 40

PE

s red

uctio

n (%

)

[AOT] mmol L-1

c

a b cb c a b c a b c

a c

a b c a cy

x x yx y x y

x x x

Fig. 4 e The mass fraction reduction of PEs (%) by the

systems of 40 mmol LL1 Tween 80 at 100 mmol LL1 NaCl

(T80) and of 40 mmol LL1 LS9 at 100 mmol LL1 NaCl (LS9)

mixed with various concentrations of AOT; a, b and c

represent the statistic testing ( p < 0.05) for T80 and x and

y represent the statistic testing ( p < 0.05) for LS9.

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 652

the solubilization of a polar compound increases when the

EON of the nonionic surfactant with the same hydrophobic

portion increases because the palisade area in themicelle was

enlarged. Even though the PEs compounds are likely hydro-

phobic, their structure composes of both hydrophobic

portions and hydrophilic portions as ester and hydroxyl

groups (Fig. 1) [9]; this may be expected that ester and

hydroxyl groups in their structure may be facilitated by an

increased polarity of the surfactant from increasing EON and

subsequently enhances the solubility to some extents.

Conversely, the C-chain length, which represents the hydro-

phobic part of a surfactant, tends to have less of an effect on

the solubilization of PEs. However, based on the overall effi-

ciency, the Tween group surfactants performed better due to

their more suitable structures, specifically their polysorbate

portions (see Fig. 2); these structures seemed to be more

compatible with the structures of the PEs than those of the

Dehydol group.

To compare the PEs reduction efficiency performance of

the two series of nonionic surfactants, a parameter known as

the hydrophileelipophile balance (HLB) of the surfactants was

plotted against the PEs reduction (Fig. 3(b)). The HLB value

generally indicates the property of a surfactant system: the

higher the HLB, the more the hydrophilic the surfactant. In

general, one can expect the optimumsolubilization to occur in

a system where the solvent has an HLB similar to that of the

surfactant solution [21]. Edris and Abd El-Galeel [22] concurred

with this in their study on the solubilization of fragrance oils

in a surfactant solution; they showed that the relationship

between the HLB of the surfactant and the oil was significant.

The results from our study revealed that the optimum HLB

compatibility with the PEs was approximately 15 (Fig. 3(b)).

Although the HLB values of the PEs were not evaluated in this

study, it can be predicted that the approximate HLB of the PEs

may be close to 15 [21].

3.1.2. PEs reduction by mixed surfactant systemsA mixed surfactant system containing nonionic and anionic

surfactants is considered a temperature-insensitive system

when compared to a single nonionic system. Moreover, it can

enhance the tolerance to salinity more than with a single

anionic system [20]. Therefore, in this part of the study, AOT

wasmixedwith a nonionic surfactant. According to the results

in the previous section (Table 1), T80 and LS9 at 40 mmol L�1

were selected to mix with AOT and NaCl to determine the

optimal combination of ingredients needed to remove the PEs

from the pressedmeal. The experimental conditions were the

same as described for the previous experiment.

To enhance the performance of an anionic surfactant, salt

is commonly added to the system because salt provides

electrolytes or positive charges to reduce the electrostatic

forces between the head groups of the surfactant [20,21]. In

this experiment, salt was added andmaintained at a constant

concentration of 100mmol L�1 for all concentrations of AOT. A

test of the various concentrations of AOT revealed that

5 mmol L�1 of AOT was the concentration yielding the highest

PEs reduction (Fig. 4). However, at this concentration of AOT,

the results for the systems containing either T80 or LS9 were

not significantly different from those of the single systems

( p < 0.05). From the graphs shown in Fig. 4, the single

surfactant systems for both nonionic surfactants, T80 and LS9,

performed slightly better than those of the mixed systems

containing 5 mmol L�1 AOT, even though the statistical

Table 2 e The mass fraction of PEs reduction (%) from theinitial pressed meal and PEs remaining in the residualmeal by the selected 4 systems of single and mixedsurfactants.

Systems PEs reduction(%)

PEs remaining inresidual meal

(mg g�1)

D.I. water 22.49 1.12

Single surfactant

(1) 40 mmol L�1 T80 81.43 0.27

(2) 40 mmol L�1 LS9 81.87 0.26

Mixed surfactants

(3) 40 mmol L�1 T80 and

5 mmol L�1 AOT at

100 mmol L�1 NaCl

81.23 0.27

(4) 40 mmol L�1 LS9 and

5 mmol L�1 AOT at

100 mmol L�1 NaCl

78.85 0.31

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 6 53

relevance of the difference was not significant ( p < 0.05). The

PEs mass fraction reduction efficiency by the T80 system

without and with 5 mmol L�1 AOT was 72.32% and 70.59%,

respectively. While the PEs mass fraction reduction efficiency

by the LS9 system without and with 5 mmol L�1 AOT was

64.76% and 62.18%, respectively. Nonetheless, it was found

that the residual meal was muchmore easily filtered from the

surfactant solution in the mixed surfactant systems than it

was in the single nonionic surfactant systems. Consequently,

the two mixed surfactant systems, 40 mmol L�1 T80 with

5 mmol L�1 AOT and 40 mmol L�1 LS9 with 5 mmol L�1 AOT,

were selected for further experimentation to determine the

effects of the NaCl concentration.

3.1.3. Effects of salt on PEs reductionNaCl is an electrolyte generally used to reduce the ionic

strength of the negative ions at the hydrophilic heads of ionic

surfactants. It generally decreases the HLB of an ionic

surfactant system [21]. Once HLB of the system decreases, it is

expected that the interfacial tension will decrease and the PEs

would be detached from the residual meal. Therefore, a salt

scan can be expected to enhance the PEs reduction efficiency.

The two selected mixed surfactant solutions from the

previous experiment were evaluated to determine the optimal

concentration of NaCl. However, the result from this experi-

ment shows that the PEs reduction efficiencies are not

significantly different for whole range of NaCl concentration

(0e300 mmol L�1). The PEs mass fraction reduction efficien-

cies were ranging from 78.24% to 81.43% and 76.98 % to 78.85 %

for the systems of mixed T80 with AOT and of LS9 with AOT,

respectively. This result indicates that lower interfacial

tension may not be a major role for PEs removal from the

substrate (Jatropha meal).

It should be noted that the PEs reduction efficiency results

obtained from this experimental step were higher than those

of the previous experiments regarding the effects of the AOT

concentrations. For the T80 and AOT mixed system, the

average PEs mass removal efficiency increased by more than

10% (from 70.59% to 81.23%); the increase was even higher for

the LS9 and AOT mixed system, in which the PEs mass frac-

tion reduction efficiency increased from 62.18% to 78.85%. One

may argue the accuracy and precision of the experimental

procedure. This discrepancymay have been caused by the age

of the pressed meal. The pressed meal used in the previous

study regarding the effects of AOT on the PEs removal effi-

ciency had been kept for more than 10 months, whereas the

experiment regarding the effects of NaCl used newly pressed

meal that had been pressed only two weeks prior to the

experiment. According to Mitra [23], the organic compounds

in the older meal require a longer time to react with the solid

phase, creating a stronger bond between the chemicals. For

this reason, the PEs can be extracted from new meal more

easily than from older meal. This finding, however, will be

evaluated and confirmed in future studies.

3.2. The effects of the physical parameters on PEsreduction

To further investigate the optimum condition for PEs reduc-

tion, physical conditions of the extraction were evaluated for

the systems of single and mixed surfactants that yielded the

highest efficiency. Table 2 describes the ingredients of the

surfactant solutions, their PEs mass fraction reduction effi-

ciencies, and the levels of the PEs that remained in the meal

after the removal process. D.I. water was also used as the PEs

washing solution under the same conditions. The PEs reduc-

tion results of the four selected systems did not differ signif-

icantly ( p < 0.05). While the mixed surfactant systems did not

remove more PEs than the single surfactant systems did,

however the mixed surfactant systems facilitated the sepa-

ration of the residual meal as previously mentioned. Two

physical parameters evaluated in this study were the contact

time and the solideliquid ratio.

3.2.1. Contact timeIn the previous experiments, the contact time was 30 min to

ensure that the meal and the surfactant were thoroughly

mixed together. However, to evaluate the optimum condi-

tions, the contact time was varied from 2 min to 30 min. Only

the mixed T80 and AOT solution system was utilized in this

study. The results in Fig. 5 clearly demonstrate that 15 min

was the optimum amount of time for mixing themeal and the

solution. Our statistical analysis showed that the removal of

PEs after 15 min (at 20 and 30 min) did not differ significantly

from the PE removal at 15 min ( p < 0.05). This result indicates

that 15 min of contact time was sufficient for the meal and

solution to properly mix.

3.2.2. Solideliquid ratio and double extractionThe solideliquid ratios were evaluated in this experiment by

varying the ratios of meal (g) and surfactant solution (L); the

ratios usedwere 50, 75, 100, 150, and 200 g L�1using the system

of mixed 40 mmol L�1 LS9 and 5 mmol L�1 AOT with

100 mmol L�1 NaCl at 15 min contact time. However, it should

be noted that the experiment conducted in this part using

10 mL of the surfactant solutions with the portion of solid

meal asmentioned earlier. In addition, other two experiments

were carried out at the contact time of 15, 20 and 30min at the

solideliquid ratios of 100, 150, and 200 g L�1. The lower ratios

50

55

60

65

70

75

80

85

90

95

100

0 5 10 15 20 25 30 35

PE

s red

uctio

n (%

)

Contact time (min)

Fig. 5 e Effects of contact time on the mass fraction

reduction of PEs (%) by the system of mixed 40 mmol LL1

Tween 80 and 5 mmol LL1 AOT at 100 mmol LL1 NaCl.

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 654

were expected to provide more space for the meal to contact

the surfactant monomers and hence reduce the interfacial

tension between the solids and the PEs. This mechanism

allowed PEs to detach and solubilize into the surfactant

solution. As expected, at the lowest solideliquid ratio yielded

the highest reduction efficiency. The same trend was found

for all sets of the experiment with the different contact times

(Fig. 6). When the ratios were the same, a contact time greater

than 15 min did little to enhance the efficiency; only a slight

increase in the PEs removal was observed. These results were

similar to those of the previous experiment, which found

15 min to be sufficient. However, the solideliquid ratio

exhibited a significant effect ( p < 0.05) on reduction of PEs.

Another approach for evaluation of the PEs reduction effi-

ciency was performed by double extraction. The same meal

was extracted twice with a fresh surfactant solution. For this

process, 2 g of meal was extracted with 20 mL of surfactant

solution (solideliquid ratio at 100 g L�1) for 15min and allowed

to precipitate for 30 min. The clear solution was then

60

65

70

75

80

85

90

0 50 100 150 200 250

PE

s red

uctio

n (%

)

Solid:Liquid ratio (g L-1

)

15 min 20 min 30 min

Fig. 6 e The mass fraction reduction of PEs (%) by the

system of mixed 40 mmol LL1 LS9 and 5 mmol LL1 AOT

with100 mmol LL1 NaCl at different solideliquid ratios and

various contact times.

decanted, and another 10 mL of the fresh surfactant solution

was added; the same extraction procedure was then per-

formed. The double extraction results are shown in Fig. 7. The

four selected surfactant solutions were evaluated in this

manner. The results show that double extraction was able to

significantly improve themass fraction reduction of PEs for all

of the systems ( p < 0.05). However, if compare to the single

extraction, the double extraction was performed under the

conditions of the total 30 min contact time with an average of

66.67 g L�1 solideliquid ratio. As compared to the result in

Fig. 6 the double extraction does not help to enhance the PEs

reduction efficiency.

3.3. Scaling up the PEs removal process

To determine the optimum conditions for the four selected

systems, the experimental scale was expanded from 1 g of

meal per 20 mL of the surfactant solution (solideliquid ratio

50 g L�1) in a 40 I-CHEM test tube to 8 g of meal per 160 mL of

solution in a 250 mL Erlenmeyer flask. Meanwhile, the

removal conditions remained constant (i.e., 31.42 rad s�1 and

15 min of contact time). Interesting results were observed; in

all cases, the larger-scale experiments yielded higher PEs

removal efficiencies (Fig. 8). This may be attributed to the

mass transfer factor [23] because, in contrast to the test tube,

the larger Erlenmeyer flask allowed the components to be

better mixed together, resulting in more coalescence

among all of the components in the systems. This result

indicates that larger-scale applications of this procedure are

promising.

3.4. Crude protein in the residual meal

Protein is an essential component in residual meal because it

can enhance the value of themeal by giving it additional uses.

Therefore, the crude protein in the pressed meal and the

detoxified meal from the four surfactant solutions was

a b80.28

b81.52 a

79.87a

79.80

d86.06

d86.06 c

83.52c

83.74

50556065707580859095

100

(1) (2) (3) (4)

PE

s red

uctio

n (%

)

System

Fig. 7 e The mass fraction reduction of PEs (%) by single

and double extractions using four selected systems: (1)

40 mmol LL1 Tween 80, (2) 40 mmol LL1 Tween 80, (3)

mixed of 40 mmol LL1 Tween and 5 mmol LL1 AOT with

100 mmol LL1 NaCl, and (4) mixed of 40 mmol LL1 Dehydol

LS9 and 5 mmol LL1 AOT with 100 mmol LL1 NaCl; aed

represent the statistic testing ( p < 0.05).

b 83.39

b 84.04

a 79.07

c 87.32

c 86.64

d 89.94

b 82.47

c 87.61

50

55

60

65

70

75

80

85

90

95

100

(1) (2) (3) (4)

PE

s re

du

ctio

n (%

)

System

Small-scale Up-scale

Fig. 8 e Comparison of the mass fraction reduction of PEs

(%) of the small-scale and up-scale experiments by the four

selected systems: (1) 40 mmol LL1 Tween 80, (2)

40 mmol LL1 Tween 80, (3) mixed of 40 mmol LL1 Tween

and 5 mmol LL1 AOT with 100 mmol LL1 NaCl, and (4)

mixed of 40 mmol LL1 Dehydol LS9 and 5 mmol LL1 AOT

with 100mmol LL1 NaCl; aed represent the statistic testing

( p < 0.05).

b i om a s s a n d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 6 55

measured by Kjeldahl’s method. The results are shown in

Table 3. The loss of crude protein tended to be higher in the

single surfactant systems and the systems that contained T80

seemed to generate higher losses of crude protein than those

with LS9 (T80 > single LS9y mixed T80 and AOT >mixed LS9

and AOT). The loss of crude protein can occur via the solubi-

lization mechanism. This indicates that the crude protein

from the residual meal tended to solubilize more in the

systems with T80 because the composition of different amino

acids makes all proteins polymeric, and they possess

a complicated structure that is more compatible with the

structure of T80. However, in the mixed systems, the lipo-

philic surfactant (AOT) may be incompatible with crude

protein and therefore reduce its solubilization in the systems

with T80 and LS9; this would explain the decreased loss of

crude protein.

According to the FAO standards [24], J. curcas seeds contain

high nutrient protein and have essential amino acids.

However, as mentioned earlier, toxins such as trypsin

Table 3 e Crude protein contents in the initial pressedmeal and residual meal after phorbol esters reduction bysurfactant solutions.

Sample Crude proteincontent (g kg�1)

Mass fractionloss of crudeprotein (%)

Initial pressed meal 179.4 e

After PEs extraction by

C Single T80 meal 144.6 19.40

C Single LS9 meal 151.0 15.83

C Mixed T80 meal 152.1 15.22

C Mixed LS9 meal 162.4 9.48

inhibitors, saponins, phytate, lectin, tannins, and PEs are

present in varying amounts. With the exception of the PEs,

these toxins can be destroyed by a moist heat treatment

[3e5,25]. Because the PEs are thermal-persistent, these

compounds have become a major concern for those wanting

to utilize J. curcas meal. Therefore, the further use of the

residual meal as a raw material for feedstock would be

impossible without the application of a detoxification process.

Aregheore et al. [10] found that a traditional heat treatment

could inactivate lectin but not the PEs. However, a heat

treatment using a temperature of 121 �Cwith 66%moisture for

30 min followed by washing the meal 4 times with 92%

methanol was able to remove 95% by mass fraction of the PEs

from the residue meal (from 1.78 mg g�1 to 0.09 mg g�1).

Martınez-Herrera et al. [25] found that meal washed with

ethanol and subsequently with 0.07% NaHCO3 decreased the

PEs to 0.08mg g�1. Nokkaew et al. [11] found that washingwith

2e3% potassium hydroxide followed with 95% ethanol over-

night can reduce the PEs from pressed meal to as low as

0.11 mg g�1, similar as the content found in a non-toxic J.

curcas variety [5].

In this study, the pressed meal PEs content of 1.45 mg g�1

was reduced to 0.20e0.30 mg g�1 depending on the surfactant

solution used. The overall mass fraction reduction efficiency

ranged from 79% to almost 90%. Although the use of surfac-

tant solutions did not lower the levels of PEs in themeal to the

level as low as those found in the non-toxic variety, the PEs

removal by a surfactant aqueous-based solution is considered

to be a clean technology. In addition, by this approach the

removed PEs in the surfactant solution can be recovered for

further uses.

4. Conclusion

The results of this study indicate that surfactant technology

provides an opportunity to develop a promising technique for

reducing the PEs from Jatropha meal. Although the concen-

trations of the remaining PEs after the removal process by

surfactant aqueous-based solutions were not as low as those

found in the non-toxic Mexican variety (0.11 mg g�1), the

highest reduction efficiency was still close to 90%. To select

the most suitable formulation of the surfactant solution for

the reduction of PEs, it was necessary to consider several

criteria. Because the PEs reduction efficiencies of the

four selected formulas did not differ significantly, other

criteria such as protein loss, the practicality of the procedure,

and the cost of the surfactant may need to be evaluated in the

future.

Acknowledgments

This work was supported by the Higher Education Research

Promotion and National Research University Project of

Thailand, Office of the Higher Education Commission, the

Center of Excellence for Environmental and Hazardous Waste

Management, Chulalongkorn University (EHWM) and PTT

Chemical Public Co, Ltd. Thailand (PTTCH), and the Graduate

School of Chulalongkorn University.

b i om a s s an d b i o e n e r g y 4 5 ( 2 0 1 2 ) 4 8e5 656

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