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Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: Kinetic and mass transfer studies
Accepted Manuscript Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: Kinetic and mass transfer studies
Sing Chuong Chuo, Akil Ahmad, Siti Hamidah Mohd-Setapar, Sarajul Fikri Mohamed, Mohd. Rafatullah PII: DOI: Reference:
S0167-7322(18)35157-2 https://doi.org/10.1016/j.molliq.2018.11.138 MOLLIQ 10048
To appear in:
Journal of Molecular Liquids
Received date: Revised date: Accepted date:
6 October 2018 15 November 2018 27 November 2018
Please cite this article as: Sing Chuong Chuo, Akil Ahmad, Siti Hamidah Mohd-Setapar, Sarajul Fikri Mohamed, Mohd. Rafatullah , Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: Kinetic and mass transfer studies. Molliq (2018), https://doi.org/10.1016/j.molliq.2018.11.138
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Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: kinetic and Mass Transfer studies Sing Chuong Chuoa, Akil Ahmada,c, Siti Hamidah Mohd-Setapara*, Sarajul Fikri Mohamedb, Mohd. Rafatullah*c a
School of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310
Department of Quantity Surveying, Faculty of Built Environment, Universiti Teknologi
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b
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UTM Skudai, Johor, Malaysia
Malaysia, 81310 UTM Skudai, Johor, Malaysia
School of Industrial Teknologi, Universiti Sains Malaysia, Penang-11800, Malaysia
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c
*Corresponding authors:
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E-mail: [email protected] (SHMS); Tel.: +60 75535496; Fax: +60 75581463.
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Email: [email protected]; [email protected] (MR)
Abstract
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Reverse micelle extraction of erythromycin and amoxicillin were studied by using eco-friendly sophorolipids biosurfactant. Application of biosurfactant can further
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improve reverse micelle extraction in terms of sustainability and environmental friendliness compared to synthetic surfactants. The mass transfer behavior of the
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antibiotics during reverse micelle extraction was investigated. Experimental results show that the reverse micelle extraction of amoxicillin and erythromycin were completed
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within 200 s. The range of combined mass transfer coefficients obtained is 1.748 × 10-8 to 1.064 × 10-7 for reverse micelle extraction of amoxicillin and 3.395 × 10-7 to 1.131 × 10-6
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for erythromycin. The rate limiting steps for each extraction process were identified. The overall mass transfer coefficients of backward extraction were found to be lower than that of forward extraction for both antibiotics which indicates that the forward extraction process was more efficient and faster than the backward extraction. Comparisons between erythromycin and amoxicillin showed that erythromycin has better equilibrium partitioning and larger calculated overall mass transfer coefficients compared to amoxicillin. This may be due to some differences in behaviors and characteristics of amoxicillin and erythromycin during the reverse micelle extraction process. This reverse
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micelle extraction method was found to be more efficient in extracting erythromycin compared to amoxicillin.
Keywords: Amoxicillin; Biosurfactant; Erythromycin; Reverse Micelles; Sophorolipids
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Introduction
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Reverse micelle extraction is a liquid-liquid extraction method utilizing reverse
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micelles formed by various surfactants for separation of organic and inorganic substances.
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This method has high selectivity, low energy consumption, and mild thermal operating conditions [1-4]. During the extraction process, biomolecules are encapsulated in reverse
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micelles and therefore avoid denaturation by direct contact with organic solvent [5]. Thus recovery of biomolecules can be enhanced by using reverse micelle extraction. The
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organic solvents and surfactants can be easily recovered and reused to reduce process costs [6]. It also has the potential for large scale downstream processing of biomolecules
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from fermentation broth [7]. Furthermore, a sustainable and environmental friendly separation method for biomolecules can be achieved through reverse micelle extraction
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utilizing biosurfactant such as sophorolipids [8]. Various biomolecules such as bromelain [9,10], soybean protein [11], bovine serum albumin [12], ovalbumin [13], lectin [14],
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penicillin G [15], lipase [16], laccase [17], adenine [18] and β-glucosidase [19] were effectively recovered using reverse micelle extraction. However, most of these studies are
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not extended to include kinetic studies during the reverse micelle extraction process. Models are very useful in studying the behavior of a system or process. For
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reverse micelle extraction, models can be used to investigate the partitioning of molecules involved, mass transfer rate, and extraction performance of the system. Kinetic studies is important in understanding the fundamentals of the physical processes occurred during the extraction for process design [20]. Through the developed models, rate limiting steps during the reverse micelle extraction process can be determined. The behavior of solutes in reverse micelle extraction is usually associated with the resistances encountered by the solutes near the interfaces of two liquids. Nishiki et al. [21] studied the mass transfer rate of lysozyme between KCl aqueous phase and AOT/isooctane solution. Their study shows that forward extraction of lysozyme is
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controlled by the diffusion in aqueous phase and solubilization process, while backward extraction is mainly controlled by the releasing process. Backward extraction is determined to be slower than forward extraction. Dövyap et al. [20] studied the mass transfer of L-isoleucine from NaOH aqueous solution to Aliquat-336/1-decanol/isooctane solution. They concluded that the rate limiting steps for this process are diffusion in aqueous phase and solubilization process. Another mass transfer study conducted by
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Mohd-Setapar et al. [22] on the mass transfer of penicillin G between KCl aqueous phase
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and dioleyl phosphoric acid (DOLPA)/isooctane solution reveals that rate limiting step in
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forward extraction is diffusion in organic phase while in backward extraction is diffusion in aqueous phase.
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There are very few studies reported on reverse micelle extraction of biomolecules using biosurfactants as compared to reverse micelle extraction using synthetic
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surfactants such as bis(2-ethylhexyl) sodium sulfosuccinate (AOT), sodium dodecyl sulfate (SDS), and cetylmethylammonium bromide (CTAB). Biosurfactants have the
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advantages of biodegradability, high surface activity, low critical micelle concentration, low toxicity, good antiviral activity, and resistance to pH, temperature and salinity
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changes [23, 24]. Peng et al. [17] showed the potential of rhamnolipids biosurfactant to extract laccase from C. versicolor. Another study showed the potential of rhamnolipids
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mixed with non-ionic surfactants for the extraction of cellulase from aqueous solution [25]. The use of biosurfactant provides a more environmental friendly operation.
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Sophorolipids is a biosurfactants which is commonly produced from nonpathogenic yeast Candida bombicola and consists of lactonic (non-ionic) form and acidic
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(anionic) form [26-28]. The biosurfactant is produced from renewable sources and has the advantages of readily biodegradable and low toxicity [29]. The properties of sophorolipids are reported to be comparable or even better than synthetic surfactants [30]. Therefore, it has the potential to be used as a sustainable and environmental friendly alternative for chemical surfactants. Downstream processing of antibiotics commonly involves solvent extraction. However, conventional solvent extraction method has the difficulties of stable emulsion hindering separation process, limited solvent choices, and long separation time [31]. Reverse micelle extraction is a potential alternative for the extraction of antibiotics. In
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this study, mass transfer of amoxicillin and erythromycin during reverse micelle extraction process was investigated. Sophorolipids biosurfactant was used to form the reverse micelles. The main purpose of this study is to examine the behaviors of antibiotics during forward and backward extraction under different solution pH. Amoxicillin and erythromycin are two commonly used antibiotics from different antibiotic classes. The differences in their properties may affect their mass transfer during
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the reverse micelle extraction. The mass transfer coefficients and rate limiting steps
Material and Method
2.1
Material Amoxicillin
trihydrate
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2.0
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during the reverse micelle extraction of antibiotics were also identified.
(C16H19N3O5S3H2O,
419.45
g/mol)
and
pure
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erythromycin A (C37H67NO13, 733.94 g/mol) were purchased from bio-WORLD, USA. Sophorolipids, potassium chloride (KCl), hydrochloric acid (HCl), sodium hydroxide
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(NaOH), and isooctane were purchased from Sigma-Aldrich, USA. All materials were of analytical grade and used without further purification. Demineralized water and isooctane
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were used to prepare the aqueous and organic phases throughout the study. Two distinct phases were obtained because isooctane and water are completely immiscible. The pH of
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aqueous solutions was adjusted by using HCl and NaOH solutions. KCl was used to adjust the ionic strength of aqueous solutions. The reverse micelle extraction was
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conducted with the help of magnetic stirrer. The measurement of antibiotic
Japan.
2.2
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concentrations was done using UV-vis spectrophotometer model UV-1800 Shimadzu,
Method
Firstly, the standard solutions of aqueous phase and organic phase were prepared. Aqueous solutions with pH range 4–10 were prepared using demineralized water and HCl or NaOH solutions. Various studies reported the production of amoxicillin at around 4 g/L within 5 h of operation [32, 33]. Since reverse micelle extraction is a fast process and able to process that amount of antibiotics within 5 h, thus 4 g/L was chosen as the starting antibiotic concentration for this study. 4.0 g/L of amoxicillin or erythromycin and 50 mM
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of KCl were dissolved in the aqueous phase to obtain the feed phase for forward extraction. 50 mM or 150 mM of KCl was dissolved in the aqueous phase to obtain the stripping phase for backward extraction. 0.3 g/L or 0.7 g/L of sophorolipids was dissolved in isooctane to obtain the reverse micellar phase for the reverse micelle extraction. The reverse micellar phase was prepared right before experiment to prevent solvent loss through vaporization. The values of parameters used in this study were all
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predetermined through previous experiments to identify the optimum reverse micelle
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extraction of amoxicillin and erythromycin [34].
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For mass transfer study of amoxicillin, forward extraction was conducted at aqueous phase pH 4.0, 5.0, 6.0, sophorolipids concentration at 0.70 g/L, KCl
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concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. On the other hand, backward extraction was conducted at feed phase pH 3.0, stripping phase pH 5.0,
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6.0, 7.0, sophorolipids concentration at 0.70 g/L, feed phase KCl concentration at 50.0 mM, stripping phase KCl concentration at 150.0mM, and 1:1 aqueous phase to organic
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phase ratio. A small, equal volume of aqueous phase and organic phase were drawn at extraction time 5 s, 30 s, 60 s, 90 s, 5 min, 15 min, and 25 min for UV spectrophotometry
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analysis. The corresponding wavelengths for maximum absorbance were 208 nm for amoxicillin and 193 nm for erythromycin.
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For mass transfer coefficient study of erythromycin, forward extraction was conducted at aqueous phase pH 8.0, 9.0, 10.0, sophorolipids concentration at 0.30 g/L,
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KCl concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. Backward extraction was conducted at feed phase pH 8.0, stripping phase pH 8.0, 9.0, 10.0,
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sophorolipids concentration at 0.30 g/L, feed phase KCl concentration at 50.0 mM, stripping phase KCl concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. An equal volume of aqueous phase and organic phase are drawn at extraction time 5 s, 30 s, 60 s, 90 s, 5 min, 15 min, and 25 min for UV spectrophotometry analysis.
2.3.
Mass Transfer Model During forward extraction, antibiotic molecules were transferred from aqueous
phase into the organic phase through reverse micelles formed by sophorolipids. Since no chemical reactions are involved during the extraction process, concentrations of
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antibiotics in aqueous phase and in organic phase at any time can be expressed by Equation 1:
VaqCaq,i = VaqCaq + VorgCorg Antibiotic concentration in aqueous phase
Caq,i
=
Initial antibiotic concentration in aqueous phase
Corg
=
Antibiotic concentration in organic phase
Vaq
=
Aqueous phase volume
Vorg
=
Organic phase volume
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=
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Where Caq
(1)
Volumes of aqueous phase and organic phase are assumed to be constant
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throughout the extraction process. After the two phases are mixed, equilibrium is achieved at certain time and the relative antibiotic concentrations can be described by
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Equation 2 for the purpose of modeling.
Caq*
= =
Equilibrium antibiotic concentration in aqueous phase Equilibrium antibiotic concentration in organic phase
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Corg* =
Equilibrium partition coefficient for forward extraction
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Where mf
(2)
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mf = Caq* / Corg*
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The value of mf is assumed to be constant under the experimental conditions investigated. Antibiotic molecules are transferred from bulk aqueous phase through boundary films of both phases into bulk organic phase. Assuming the resistance of interfacial solubilization is negligible, the transfer rate equations are given in the following equations. Jf = kaq,f (Caq – Caq,in)
(3)
Jf = korg,f (Corg,in – Corg)
(4)
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Where Jf kaq,f
=
Flux of antibiotics in forward extraction
=
Aqueous film mass transfer coefficient during forward extraction
korg,f
=
Organic film mass transfer coefficient during forward extraction Antibiotics concentration at aqueous film interface
Corg,in =
Antibiotics concentration at organic film interface
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=
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Caq,in
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Assuming that the extraction process is at steady state and antibiotic concentrations of both films is in equilibrium at the interfaces, the transfer rate equations
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Jf = Kf (Caq – mfCorg) = – (Vaq /A) (dCaq / dt)
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are equal and the following equations are obtained:
(6)
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1/Kf = 1/kaq,f + mf/korg,f
(5)
=
Overall mass transfer coefficient during forward extraction
A
=
Interfacial area
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Where Kf
In this study, the actual value of A is unknown hence only the combined mass
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transfer coefficient, Kf A can be obtained. Unit for combined mass transfer coefficient is
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L/s. Manipulating Equation 5 to obtain:
(7)
1/KfA = 1/kaq,f A + m/korg,f A
(8)
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(Vaq) (dCaq / dt) = - Kf A (Caq – mfCorg)
Substitute for Corg in Equation 7 using Equation 1 and simplify to obtain following equations: dCaq / dt = – αf (Caq – βfCaq,i)
(9)
αf = Kf A/Vaq (1+ mfVr)
(10)
βf = mfVr/(1+ mfVr)
(11)
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The term Vr is the phase volume ratio (Vaq / Vorg) and its value for this study is always equal to 1. Integrating Equation 9 from t = 0 to t = t, following equation is obtained: ln|(Caq – βfCaq,i)/(1 – βf) Caq,i | = – αf t
(12)
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Equation 12 suggests that plotting ln|(Caq – βfCaq,i)/(1 – βf) Caq,i | against t will
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yield a straight line with gradient – αfthat passes through the origin. Concentration of
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antibiotics at time t during extraction can also be calculated from the equation. During backward extraction, extraction conditions were adjusted to favor the
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transfer of antibiotic molecules from reverse micellar phase into fresh aqueous phase to recover them. The kinetic model development is similar to those for forward extraction.
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The concentrations of antibiotics in aqueous phase and in organic phase at any time with
=
Initial antibiotic concentration in organic phase
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Where Corg,i
(13)
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VorgCorg,i = VaqCaq + VorgCorg
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no chemical reaction involved can be expressed by Equation 13:
Volumes of both phases are assumed to be constant throughout the extraction
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process. After the two phases are mixed, equilibrium is achieved at certain time and the
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relative antibiotic concentrations are given by Equation 14:
mb = Corg* / Caq* Where mb
=
(14)
Equilibrium partition coefficient for backward extraction
The value of mb is assumed to be constant under the experiment conditions investigated. Assuming the resistance of interfacial release of antibiotic molecules is negligible, the transfer rate equations are given as followed:
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Jb = kaq,b (Caq,in – Caq)
(15)
Jb = korg,b (Corg – Corg,in)
(16)
Where Jb kaq,b
=
Flux of antibiotics in backward extraction
=
Aqueous film mass transfer coefficient during backward extraction
=
Organic film mass transfer coefficient during backward
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korg,b
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extraction
Assuming the extraction process is at steady state and antibiotic concentrations of
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both films are in equilibrium at the interfaces, the transfer rate equations are equal and
(Vorg) (dCorg / dt) = - Kb A (Corg – mbCaq)
=
(18)
Overall mass transfer coefficient during backward extraction
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Where Kb
(17)
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1/KbA = mb/kaq,b A + 1/korg,b A
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following equations are obtained:
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The actual value of A is unknown hence only the combined mass transfer coefficient, Kb A can be obtained. Unit for combined mass transfer coefficient is L/s.
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Substitute for Caq in Equation 17 using Equation 13, simplify and integrate from t = 0 to t
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= t to obtain following equations: ln|(Corg – βbCorg,i)/(1 – βb) Corg,i | = – αb t
(19)
αb = Kb A/Vorg (1+ mbVr)
(20)
βb = mbVr/(1+ mbVr)
(21)
The value of Vr is always equal to 1 for this study. Equation 19 indicates that plotting ln|(Corg – βbCorg,i)/(1 – βb) Corg,i | against t will yield a straight line with gradient – αb that passes through the origin.
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3.0
Result and Discussion
3.1
Forward Extraction Amoxicillin and erythromycin have insignificant solubility in isooctane without
sophorolipids. Formation of reverse micelles allows these antibiotics to be transferred into the organic phase. Therefore, this extraction method offers wider range of solvents which are safer compared to conventional solvent extraction. Our previous studies
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reported 6 g/L DOLPA for reverse micelle extraction of penicillin G and up to 102 g/L
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AOT/TWEEN 85 for extraction of amoxicillin [22, 35]. Only up to 1 g/L sophorolipids
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was used in this study. Peng et al. [17] in their study used up to 2.15 g/L rhamnolipids for reverse micelle extraction of laccase. This shows that biosurfactants can significantly
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reduce the amount of surfactants needed for reverse micelle extraction. This can make the extraction process easier to operate and reduce chemical storage needed, potentially
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reducing the process costs.
The effects of feed aqueous phase pH on the forward extraction of amoxicillin
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and erythromycin are shown in Fig. 1. The variation of antibiotic concentrations shown in Fig. 1 is typical for reverse micelle extraction of protein [36, 22]. In Figure 1(a), forward
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extraction efficiency of amoxicillin is higher at aqueous phase pH 4 and decreases when higher aqueous phase pH is used. Isoelectric point of amoxicillin is 4.7 [35]. Therefore,
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it will possess net positive charge in solution with pH less than 4.7 and on the other hand will possess net negative charge in solution with pH higher than 4.7. At aqueous phase
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pH 4, both amoxicillin and sophorolipids possess opposite net charges and thus are attracted to each other. When aqueous phase pH increased to 5 and 6, amoxicillin and
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sophorolipids are having same net charges and thus the amount of amoxicillin transferred to the reverse micellar phase gradually decreases. This indicates that electrostatic interactions between amoxicillin molecules and sophorolipids reverse micelles are significant during the forward extraction process. Similar effects of aqueous phase pH on the forward extraction of proteins are reported for tannase [37], lipase [16], and βglucosidase [19]. Fig. 1(a) also shows that forward extraction of amoxicillin is a rapid process. Amoxicillin concentration reaches a theoretical equilibrium value for each aqueous phase pH within 200 s of mixing time.
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Fig. 2 shows the application of Equation 12 for the forward extraction of amoxicillin and erythromycin. Combined mass transfer coefficients for amoxicillin are calculated from the gradients in Fig. 2(a). Individual mass transfer coefficients are obtained from Fig. 3(a) and the values are summarized in Table 1. When the feed aqueous phase pH is increased, less amoxicillin is solubilized into the organic phase and the transfer rate also decreases. This is due to the diminishing attractive electrostatic
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interactions between amoxicillin molecules and sophorolipids reverse micelles at pH
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higher than pI of amoxicillin. The calculated values of korg,f A is larger than kaq,f A
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indicating that the rate limiting step for the forward extraction of amoxicillin is diffusion process in aqueous phase.
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Fig. 1(b) shows the effect of feed aqueous phase pH on the forward extraction of erythromycin. The combined mass transfer coefficients for erythromycin are calculated
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from the gradients in Fig. 2(b). Individual mass transfer coefficients are obtained from Fig. 3(b) and the values are summarized in Table 2. Forward extraction of erythromycin
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is also a rapid process as shown in Fig. 1(b). Theoretical equilibrium is reached within 100 s for all aqueous phase pH tested. The calculated values of combined mass transfer
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coefficients shows that the forward extraction rate decreases at higher aqueous phase pH. Erythromycin molecules net positive charge at solution pH below 8.6 [38], thus having
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stronger attractive electrostatic interactions with sophorolipids head groups, allowing more erythromycin molecules to be solubilized into the reverse micellar phase. When
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feed phase pH is further increased to above 8.6, erythromycin molecules mostly exist in non-polar form. Therefore, the attractive electrostatic interactions diminished and the
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mass transfer rates become lower. The calculated values of kaq,f A is significantly larger than korg,f A which indicates that the forward extraction of erythromycin is limited by the diffusion process in organic phase.
3.2
Backward Extraction The effects of stripping aqueous phase pH on the amount of amoxicillin and
erythromycin recovered to the aqueous phase are shown in Fig. 4. Fig. 4(a) shows that amoxicillin recovery is the lowest at stripping aqueous phase pH 5 because it is very close to the pI of amoxicillin, thus the attractive interactions are stronger compared to
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those at higher pH value. Amoxicillin recovery increases at pH 6 due to the increasing repulsive electrostatic interactions between amoxicillin molecules and sophorolipids. At aqueous phase pH 7, amoxicillin recovery decreases even though the repulsive electrostatic interactions should be higher than those at pH 6. This is due to the degradation of amoxicillin in neutral and alkaline medium [35]. Fig. 4(a) also shows that the theoretical equilibrium is achieved within 200 s of extraction time. This indicates that
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the backward extraction can be a fast process.
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Fig. 5 shows the application of Equation 19 for the backward extraction of
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amoxicillin and erythromycin. Combined mass transfer coefficients for amoxicillin are calculated from the gradients in Fig. 5(a). Individual mass transfer coefficients are
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obtained from Fig. 6(a) and the values are summarized in Table 3. The mass transfer rate increases when stripping aqueous phase pH increases from 5 to 6. This is due to the
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stronger repulsive electrostatic interactions between amoxicillin molecules and sophorolipids at solution pH higher than pI of amoxicillin. Data in Fig. 6(a) scatter far
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away from a linear regression suggests that the mass transfer model may not be suitable to describe the backward extraction of amoxicillin at this pH range. This may due to the
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degradation of amoxicillin at stripping aqueous phase pH 7. Since one of the assumptions is that no chemical reaction is involved during the extraction process, the degradation of
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amoxicillin causes the calculated KbA for pH 7 to be not consistent with the calculated KbA for pH 5 and pH 6. Same inference is true for the mb values. Nevertheless, attempt
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on linear regression analysis shows that korg,b A is smaller than kaq,b A. Backward extraction of amoxicillin may be restricted by the diffusion process in organic phase.
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Fig. 4(b) shows the effect of stripping aqueous phase pH on the amount of erythromycin recovered to the aqueous phase. Backward extraction of erythromycin is a fast process as seen in Fig. 4(b) where the theoretical equilibrium is achieved within 200 s of extraction time. The increasing amount of erythromycin recovered when aqueous phase pH is increased from 8 to 10 is due to diminishing electrostatic interactions between erythromycin and sophorolipids, thus allowing more erythromycin molecules to be released into stripping phase [34]. Gaikaiwari et al. [37] also reported that maximum backward extraction efficiency is achieved at pH with minimum electrostatic interactions between biomolecules and reverse micelles.
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The combined mass transfer coefficients for erythromycin are calculated from the gradients in Fig. 5(b). Individual mass transfer coefficients are obtained from Fig. 6(b) and the values are summarized in Table 4. The mass transfer rate increases when stripping aqueous phase pH is increased from 8 to 10. The weakening of electrostatic interactions between erythromycin and sophorolipids allows faster release of erythromycin molecules from organic phase to aqueous phase. The calculated values of
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erythromycin is limited by the diffusion process in organic phase.
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korg,b A is significantly lower than kaq,b A indicating that the backward extraction of
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The separation time needed for reverse micelle extraction of amoxicillin and erythromycin obtained in this study is very short compared to conventional solvent
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extraction of antibiotics using butyl acetate which can take 35 h for separation process [39]. Reverse micelle extraction of penicillin G with chemical surfactants took more than
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15 min to achieve theoretical equilibrium [22]. Application of sophorolipids may have
3.3
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enhanced the extraction rate of antibiotics.
Comparisons
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Amoxicillin and erythromycin belong to different class of antibiotics having different kind of properties. Erythromycin has more complex molecular structure and
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larger surface area compared to those of amoxicillin. These properties affect their behavior during the reverse micelle extraction process.
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The effects of aqueous phase pH on the mass transfer of amoxicillin and erythromycin during reverse micelle extraction were investigated because aqueous phase
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pH is the most dominant factor during the extraction process. Similar extraction behavior was observed when the aqueous phase pH was varied during the extraction of both antibiotics. This is due to the nature of both antibiotics to possess net negative charge at below their pI values, thus having strong attractive electrostatic interactions with the surfactant head groups. When aqueous phase pH is increased to higher than their pI values, the attractive electrostatic interactions weakened and promotes the release of antibiotics from reverse micellar phase into aqueous phase. Both antibiotics are very sensitive to the surrounding pH thus the whole extraction process should be conducted within their own pH range to avoid degradation of the antibiotics.
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The most significant difference between reverse micelle extraction of amoxicillin and erythromycin is the amount of antibiotics extracted during forward extraction and the amount of antibiotics recovered during backward extraction. Large mf and mb values obtained for the extraction of amoxicillin indicate that only small amount of amoxicillin is extracted or recovered under the extraction conditions used in this study. On the other hand, large amount of erythromycin can be extracted during forward extraction and
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satisfactory amount of erythromycin can be recovered during backward extraction.
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Different sizes, molecular structures, and nature of the antibiotics should be the reason
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for this observation. The larger size and complex structure of erythromycin molecules may have allowed more interactions with the reverse micelles. The overall mb values in
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this study are larger than mf values, showing that the backward extraction is more difficult compared to forward extraction. This is due to the strong attractive interactions
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between antibiotic molecules and sophorolipids head groups as well as stability of reverse micelles.
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Theoretical equilibriums were achieved within 200 s for the reverse micelle extraction of both antibiotics. By further processing the mixture with fast phase
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separation equipment such as centrifuge, the antibiotic separation process can be completed in short time. The lower overall mass transfer coefficients of backward
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extraction compared to that of forward extraction been calculated shows that the backward extraction of antibiotics is more difficult than the forward extraction process.
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Reverse micelle extraction of erythromycin give a larger calculated overall mass transfer coefficients compared to amoxicillin, indicating that the extraction conditions examined
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in this study are more efficient for the separation of erythromycin than that of amoxicillin. Analysis on individual mass transfer coefficients shows that the reverse micelles extraction of erythromycin is mostly governed by the diffusion process in organic phase. On the other hand, the rate limiting step during forward extraction of amoxicillin is the diffusion process in aqueous phase. The rate limiting step during backward extraction of amoxicillin cannot be confirmed because the linear regression does not fit the data very well. Nevertheless, this observation suggests that there may be some differences on how amoxicillin and erythromycin molecules are solubilized into the organic phase or released into the stripping aqueous phase.
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4.0
Conclusion Mass transfer models to describe the reverse micelle extraction behavior of
antibiotics have been successfully developed and tested. The reverse micelle extraction is pH-dependent process. The forward and backward extractions of both antibiotics are found to be fast processes. Theoretical equilibriums were achieved within 200 s of
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extraction time for all cases. The rate limiting step during forward extraction of
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amoxicillin is the diffusion process in aqueous phase but the rate limiting step during
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backward extraction of amoxicillin cannot be confirmed due to significant lack of fit. The diffusion process in organic phase is identified as the rate limiting step during both
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forward and backward extractions of erythromycin. Reverse micelle extraction method in this study gives better performance for the separation of erythromycin compared to
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amoxicillin. This indicates that different properties possessed by different antibiotics have significant influence on their interactions with the reverse micelles, thus affecting
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the outcomes of the reverse micelle extraction. Reverse micelle extraction has the potential to be applied in downstream processing of antibiotics. It can significantly save
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the cost and time of the separation of antibiotics. Furthermore, using biosurfactants such
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Acknowledgment
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as sophorolipids can ensure that the process is environmental friendly.
The authors thank the financial supports from Research Management Center
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(RMC), grant no. (Q.J130000.2546.17H02), UniversitiTeknologi Malaysia (UTM).
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Figure Captions:
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Fig. 1. Forward extraction of (a) amoxicillin and (b) erythromycin at different feed
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aqueous phase pH
Fig. 2. Forward extraction rates of (a) amoxicillin and (b) erythromycin at different feed
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aqueous phase pH
Fig. 3. Mass transfer coefficients for forward extraction of (a) amoxicillin and (b)
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erythromycin
Fig. 4. Recovery of (a) amoxicillin and (b) erythromycin at different stripping aqueous
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phase pH
Fig. 5. Backward extraction rates of (a) amoxicillin and (b) erythromycinat different
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stripping aqueous phase pH
Fig. 6. Mass transfer coefficients for backward extraction of (a) amoxicillin and (b)
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Table Legends:
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erythromycin
Table 1. Values of equilibrium partition coefficient and mass transfer coefficients for the
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forward extraction of amoxicillin at different feed aqueous phase pH Table 2. Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of erythromycin at different feed aqueous phase pH Table 3. Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of amoxicillin at different stripping aqueous phase pH Table 4. Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of erythromycin at different stripping aqueous phase pH
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Table 1: Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of amoxicillin at different feed aqueous phase pH KfA
4
2.077
5.240 × 10-8
5
2.636
3.919 × 10-8
6
9.000
1.748 × 10-8
kaq,f A
korg,f A
1.025 × 10-7
1.890 × 10-7
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mf
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pH
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Table 2: Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of erythromycin at different feed aqueous phase pH KfA
8
0.308
1.131 × 10-6
9
0.341
8.710 × 10-7
10
0.565
5.904 × 10-7
kaq,f A
korg,f A
1.386 × 10-5
3.459 × 10-7
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mf
AC
CE
PT
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M
AN
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pH
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Table 3: Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of amoxicillin at different stripping aqueous phase pH KbA
5
5.190
3.344 × 10-8
6
2.250
1.064 × 10-7
7
6.182
5.827 × 10-8
kaq,b A
korg,b A
3.210 × 10-7
2.139 × 10-7
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mb
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PT
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AN
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pH
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Table 4: Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of erythromycin at different stripping aqueous phase pH KbA
8
2.140
3.395 × 10-7
9
1.900
3.649 × 10-7
10
1.010
4.806 × 10-7
kaq,b A
korg,b A
1.318 × 10-6
7.625 × 10-7
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mb
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pH
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Research Highlights: Eco-friendly sophorolipids biosurfactant was used in reverse micelle extraction of antibiotics Kinetic and mass transfers behavior were studied for reverse micelle extraction of
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antibiotics
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Effects of aqueous phase pH on the mass transfer was investigated for amoxicillin and erythromycin extraction
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Reverse micelle extraction of antibiotics were completed within 200 s
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Forward and backward extraction methods were investigated
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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Sing Chuong Chuo, Akil Ahmad, Siti Hamidah Mohd-Setapar, Sarajul Fikri Mohamed, Mohd. Rafatullah PII: DOI: Reference:
S0167-7322(18)35157-2 https://doi.org/10.1016/j.molliq.2018.11.138 MOLLIQ 10048
To appear in:
Journal of Molecular Liquids
Received date: Revised date: Accepted date:
6 October 2018 15 November 2018 27 November 2018
Please cite this article as: Sing Chuong Chuo, Akil Ahmad, Siti Hamidah Mohd-Setapar, Sarajul Fikri Mohamed, Mohd. Rafatullah , Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: Kinetic and mass transfer studies. Molliq (2018), https://doi.org/10.1016/j.molliq.2018.11.138
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Utilization of green sophorolipids biosurfactant in reverse micelle extraction of antibiotics: kinetic and Mass Transfer studies Sing Chuong Chuoa, Akil Ahmada,c, Siti Hamidah Mohd-Setapara*, Sarajul Fikri Mohamedb, Mohd. Rafatullah*c a
School of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310
Department of Quantity Surveying, Faculty of Built Environment, Universiti Teknologi
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b
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UTM Skudai, Johor, Malaysia
Malaysia, 81310 UTM Skudai, Johor, Malaysia
School of Industrial Teknologi, Universiti Sains Malaysia, Penang-11800, Malaysia
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c
*Corresponding authors:
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E-mail: [email protected] (SHMS); Tel.: +60 75535496; Fax: +60 75581463.
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Email: [email protected]; [email protected] (MR)
Abstract
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Reverse micelle extraction of erythromycin and amoxicillin were studied by using eco-friendly sophorolipids biosurfactant. Application of biosurfactant can further
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improve reverse micelle extraction in terms of sustainability and environmental friendliness compared to synthetic surfactants. The mass transfer behavior of the
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antibiotics during reverse micelle extraction was investigated. Experimental results show that the reverse micelle extraction of amoxicillin and erythromycin were completed
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within 200 s. The range of combined mass transfer coefficients obtained is 1.748 × 10-8 to 1.064 × 10-7 for reverse micelle extraction of amoxicillin and 3.395 × 10-7 to 1.131 × 10-6
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for erythromycin. The rate limiting steps for each extraction process were identified. The overall mass transfer coefficients of backward extraction were found to be lower than that of forward extraction for both antibiotics which indicates that the forward extraction process was more efficient and faster than the backward extraction. Comparisons between erythromycin and amoxicillin showed that erythromycin has better equilibrium partitioning and larger calculated overall mass transfer coefficients compared to amoxicillin. This may be due to some differences in behaviors and characteristics of amoxicillin and erythromycin during the reverse micelle extraction process. This reverse
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micelle extraction method was found to be more efficient in extracting erythromycin compared to amoxicillin.
Keywords: Amoxicillin; Biosurfactant; Erythromycin; Reverse Micelles; Sophorolipids
1.0
Introduction
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Reverse micelle extraction is a liquid-liquid extraction method utilizing reverse
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micelles formed by various surfactants for separation of organic and inorganic substances.
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This method has high selectivity, low energy consumption, and mild thermal operating conditions [1-4]. During the extraction process, biomolecules are encapsulated in reverse
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micelles and therefore avoid denaturation by direct contact with organic solvent [5]. Thus recovery of biomolecules can be enhanced by using reverse micelle extraction. The
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organic solvents and surfactants can be easily recovered and reused to reduce process costs [6]. It also has the potential for large scale downstream processing of biomolecules
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from fermentation broth [7]. Furthermore, a sustainable and environmental friendly separation method for biomolecules can be achieved through reverse micelle extraction
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utilizing biosurfactant such as sophorolipids [8]. Various biomolecules such as bromelain [9,10], soybean protein [11], bovine serum albumin [12], ovalbumin [13], lectin [14],
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penicillin G [15], lipase [16], laccase [17], adenine [18] and β-glucosidase [19] were effectively recovered using reverse micelle extraction. However, most of these studies are
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not extended to include kinetic studies during the reverse micelle extraction process. Models are very useful in studying the behavior of a system or process. For
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reverse micelle extraction, models can be used to investigate the partitioning of molecules involved, mass transfer rate, and extraction performance of the system. Kinetic studies is important in understanding the fundamentals of the physical processes occurred during the extraction for process design [20]. Through the developed models, rate limiting steps during the reverse micelle extraction process can be determined. The behavior of solutes in reverse micelle extraction is usually associated with the resistances encountered by the solutes near the interfaces of two liquids. Nishiki et al. [21] studied the mass transfer rate of lysozyme between KCl aqueous phase and AOT/isooctane solution. Their study shows that forward extraction of lysozyme is
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controlled by the diffusion in aqueous phase and solubilization process, while backward extraction is mainly controlled by the releasing process. Backward extraction is determined to be slower than forward extraction. Dövyap et al. [20] studied the mass transfer of L-isoleucine from NaOH aqueous solution to Aliquat-336/1-decanol/isooctane solution. They concluded that the rate limiting steps for this process are diffusion in aqueous phase and solubilization process. Another mass transfer study conducted by
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Mohd-Setapar et al. [22] on the mass transfer of penicillin G between KCl aqueous phase
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and dioleyl phosphoric acid (DOLPA)/isooctane solution reveals that rate limiting step in
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forward extraction is diffusion in organic phase while in backward extraction is diffusion in aqueous phase.
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There are very few studies reported on reverse micelle extraction of biomolecules using biosurfactants as compared to reverse micelle extraction using synthetic
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surfactants such as bis(2-ethylhexyl) sodium sulfosuccinate (AOT), sodium dodecyl sulfate (SDS), and cetylmethylammonium bromide (CTAB). Biosurfactants have the
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advantages of biodegradability, high surface activity, low critical micelle concentration, low toxicity, good antiviral activity, and resistance to pH, temperature and salinity
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changes [23, 24]. Peng et al. [17] showed the potential of rhamnolipids biosurfactant to extract laccase from C. versicolor. Another study showed the potential of rhamnolipids
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mixed with non-ionic surfactants for the extraction of cellulase from aqueous solution [25]. The use of biosurfactant provides a more environmental friendly operation.
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Sophorolipids is a biosurfactants which is commonly produced from nonpathogenic yeast Candida bombicola and consists of lactonic (non-ionic) form and acidic
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(anionic) form [26-28]. The biosurfactant is produced from renewable sources and has the advantages of readily biodegradable and low toxicity [29]. The properties of sophorolipids are reported to be comparable or even better than synthetic surfactants [30]. Therefore, it has the potential to be used as a sustainable and environmental friendly alternative for chemical surfactants. Downstream processing of antibiotics commonly involves solvent extraction. However, conventional solvent extraction method has the difficulties of stable emulsion hindering separation process, limited solvent choices, and long separation time [31]. Reverse micelle extraction is a potential alternative for the extraction of antibiotics. In
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this study, mass transfer of amoxicillin and erythromycin during reverse micelle extraction process was investigated. Sophorolipids biosurfactant was used to form the reverse micelles. The main purpose of this study is to examine the behaviors of antibiotics during forward and backward extraction under different solution pH. Amoxicillin and erythromycin are two commonly used antibiotics from different antibiotic classes. The differences in their properties may affect their mass transfer during
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the reverse micelle extraction. The mass transfer coefficients and rate limiting steps
Material and Method
2.1
Material Amoxicillin
trihydrate
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2.0
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during the reverse micelle extraction of antibiotics were also identified.
(C16H19N3O5S3H2O,
419.45
g/mol)
and
pure
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erythromycin A (C37H67NO13, 733.94 g/mol) were purchased from bio-WORLD, USA. Sophorolipids, potassium chloride (KCl), hydrochloric acid (HCl), sodium hydroxide
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(NaOH), and isooctane were purchased from Sigma-Aldrich, USA. All materials were of analytical grade and used without further purification. Demineralized water and isooctane
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were used to prepare the aqueous and organic phases throughout the study. Two distinct phases were obtained because isooctane and water are completely immiscible. The pH of
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aqueous solutions was adjusted by using HCl and NaOH solutions. KCl was used to adjust the ionic strength of aqueous solutions. The reverse micelle extraction was
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conducted with the help of magnetic stirrer. The measurement of antibiotic
Japan.
2.2
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concentrations was done using UV-vis spectrophotometer model UV-1800 Shimadzu,
Method
Firstly, the standard solutions of aqueous phase and organic phase were prepared. Aqueous solutions with pH range 4–10 were prepared using demineralized water and HCl or NaOH solutions. Various studies reported the production of amoxicillin at around 4 g/L within 5 h of operation [32, 33]. Since reverse micelle extraction is a fast process and able to process that amount of antibiotics within 5 h, thus 4 g/L was chosen as the starting antibiotic concentration for this study. 4.0 g/L of amoxicillin or erythromycin and 50 mM
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of KCl were dissolved in the aqueous phase to obtain the feed phase for forward extraction. 50 mM or 150 mM of KCl was dissolved in the aqueous phase to obtain the stripping phase for backward extraction. 0.3 g/L or 0.7 g/L of sophorolipids was dissolved in isooctane to obtain the reverse micellar phase for the reverse micelle extraction. The reverse micellar phase was prepared right before experiment to prevent solvent loss through vaporization. The values of parameters used in this study were all
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predetermined through previous experiments to identify the optimum reverse micelle
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extraction of amoxicillin and erythromycin [34].
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For mass transfer study of amoxicillin, forward extraction was conducted at aqueous phase pH 4.0, 5.0, 6.0, sophorolipids concentration at 0.70 g/L, KCl
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concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. On the other hand, backward extraction was conducted at feed phase pH 3.0, stripping phase pH 5.0,
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6.0, 7.0, sophorolipids concentration at 0.70 g/L, feed phase KCl concentration at 50.0 mM, stripping phase KCl concentration at 150.0mM, and 1:1 aqueous phase to organic
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phase ratio. A small, equal volume of aqueous phase and organic phase were drawn at extraction time 5 s, 30 s, 60 s, 90 s, 5 min, 15 min, and 25 min for UV spectrophotometry
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analysis. The corresponding wavelengths for maximum absorbance were 208 nm for amoxicillin and 193 nm for erythromycin.
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For mass transfer coefficient study of erythromycin, forward extraction was conducted at aqueous phase pH 8.0, 9.0, 10.0, sophorolipids concentration at 0.30 g/L,
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KCl concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. Backward extraction was conducted at feed phase pH 8.0, stripping phase pH 8.0, 9.0, 10.0,
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sophorolipids concentration at 0.30 g/L, feed phase KCl concentration at 50.0 mM, stripping phase KCl concentration at 50.0 mM, and 1:1 aqueous phase to organic phase ratio. An equal volume of aqueous phase and organic phase are drawn at extraction time 5 s, 30 s, 60 s, 90 s, 5 min, 15 min, and 25 min for UV spectrophotometry analysis.
2.3.
Mass Transfer Model During forward extraction, antibiotic molecules were transferred from aqueous
phase into the organic phase through reverse micelles formed by sophorolipids. Since no chemical reactions are involved during the extraction process, concentrations of
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antibiotics in aqueous phase and in organic phase at any time can be expressed by Equation 1:
VaqCaq,i = VaqCaq + VorgCorg Antibiotic concentration in aqueous phase
Caq,i
=
Initial antibiotic concentration in aqueous phase
Corg
=
Antibiotic concentration in organic phase
Vaq
=
Aqueous phase volume
Vorg
=
Organic phase volume
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=
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Where Caq
(1)
Volumes of aqueous phase and organic phase are assumed to be constant
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throughout the extraction process. After the two phases are mixed, equilibrium is achieved at certain time and the relative antibiotic concentrations can be described by
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Equation 2 for the purpose of modeling.
Caq*
= =
Equilibrium antibiotic concentration in aqueous phase Equilibrium antibiotic concentration in organic phase
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Corg* =
Equilibrium partition coefficient for forward extraction
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Where mf
(2)
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mf = Caq* / Corg*
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The value of mf is assumed to be constant under the experimental conditions investigated. Antibiotic molecules are transferred from bulk aqueous phase through boundary films of both phases into bulk organic phase. Assuming the resistance of interfacial solubilization is negligible, the transfer rate equations are given in the following equations. Jf = kaq,f (Caq – Caq,in)
(3)
Jf = korg,f (Corg,in – Corg)
(4)
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Where Jf kaq,f
=
Flux of antibiotics in forward extraction
=
Aqueous film mass transfer coefficient during forward extraction
korg,f
=
Organic film mass transfer coefficient during forward extraction Antibiotics concentration at aqueous film interface
Corg,in =
Antibiotics concentration at organic film interface
T
=
IP
Caq,in
CR
Assuming that the extraction process is at steady state and antibiotic concentrations of both films is in equilibrium at the interfaces, the transfer rate equations
AN
Jf = Kf (Caq – mfCorg) = – (Vaq /A) (dCaq / dt)
US
are equal and the following equations are obtained:
(6)
M
1/Kf = 1/kaq,f + mf/korg,f
(5)
=
Overall mass transfer coefficient during forward extraction
A
=
Interfacial area
ED
Where Kf
In this study, the actual value of A is unknown hence only the combined mass
PT
transfer coefficient, Kf A can be obtained. Unit for combined mass transfer coefficient is
CE
L/s. Manipulating Equation 5 to obtain:
(7)
1/KfA = 1/kaq,f A + m/korg,f A
(8)
AC
(Vaq) (dCaq / dt) = - Kf A (Caq – mfCorg)
Substitute for Corg in Equation 7 using Equation 1 and simplify to obtain following equations: dCaq / dt = – αf (Caq – βfCaq,i)
(9)
αf = Kf A/Vaq (1+ mfVr)
(10)
βf = mfVr/(1+ mfVr)
(11)
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The term Vr is the phase volume ratio (Vaq / Vorg) and its value for this study is always equal to 1. Integrating Equation 9 from t = 0 to t = t, following equation is obtained: ln|(Caq – βfCaq,i)/(1 – βf) Caq,i | = – αf t
(12)
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Equation 12 suggests that plotting ln|(Caq – βfCaq,i)/(1 – βf) Caq,i | against t will
IP
yield a straight line with gradient – αfthat passes through the origin. Concentration of
CR
antibiotics at time t during extraction can also be calculated from the equation. During backward extraction, extraction conditions were adjusted to favor the
US
transfer of antibiotic molecules from reverse micellar phase into fresh aqueous phase to recover them. The kinetic model development is similar to those for forward extraction.
AN
The concentrations of antibiotics in aqueous phase and in organic phase at any time with
=
Initial antibiotic concentration in organic phase
PT
Where Corg,i
(13)
ED
VorgCorg,i = VaqCaq + VorgCorg
M
no chemical reaction involved can be expressed by Equation 13:
Volumes of both phases are assumed to be constant throughout the extraction
CE
process. After the two phases are mixed, equilibrium is achieved at certain time and the
AC
relative antibiotic concentrations are given by Equation 14:
mb = Corg* / Caq* Where mb
=
(14)
Equilibrium partition coefficient for backward extraction
The value of mb is assumed to be constant under the experiment conditions investigated. Assuming the resistance of interfacial release of antibiotic molecules is negligible, the transfer rate equations are given as followed:
ACCEPTED MANUSCRIPT 9
Jb = kaq,b (Caq,in – Caq)
(15)
Jb = korg,b (Corg – Corg,in)
(16)
Where Jb kaq,b
=
Flux of antibiotics in backward extraction
=
Aqueous film mass transfer coefficient during backward extraction
=
Organic film mass transfer coefficient during backward
T
korg,b
CR
IP
extraction
Assuming the extraction process is at steady state and antibiotic concentrations of
US
both films are in equilibrium at the interfaces, the transfer rate equations are equal and
(Vorg) (dCorg / dt) = - Kb A (Corg – mbCaq)
=
(18)
Overall mass transfer coefficient during backward extraction
ED
Where Kb
(17)
M
1/KbA = mb/kaq,b A + 1/korg,b A
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following equations are obtained:
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The actual value of A is unknown hence only the combined mass transfer coefficient, Kb A can be obtained. Unit for combined mass transfer coefficient is L/s.
CE
Substitute for Caq in Equation 17 using Equation 13, simplify and integrate from t = 0 to t
AC
= t to obtain following equations: ln|(Corg – βbCorg,i)/(1 – βb) Corg,i | = – αb t
(19)
αb = Kb A/Vorg (1+ mbVr)
(20)
βb = mbVr/(1+ mbVr)
(21)
The value of Vr is always equal to 1 for this study. Equation 19 indicates that plotting ln|(Corg – βbCorg,i)/(1 – βb) Corg,i | against t will yield a straight line with gradient – αb that passes through the origin.
ACCEPTED MANUSCRIPT 10
3.0
Result and Discussion
3.1
Forward Extraction Amoxicillin and erythromycin have insignificant solubility in isooctane without
sophorolipids. Formation of reverse micelles allows these antibiotics to be transferred into the organic phase. Therefore, this extraction method offers wider range of solvents which are safer compared to conventional solvent extraction. Our previous studies
T
reported 6 g/L DOLPA for reverse micelle extraction of penicillin G and up to 102 g/L
IP
AOT/TWEEN 85 for extraction of amoxicillin [22, 35]. Only up to 1 g/L sophorolipids
CR
was used in this study. Peng et al. [17] in their study used up to 2.15 g/L rhamnolipids for reverse micelle extraction of laccase. This shows that biosurfactants can significantly
US
reduce the amount of surfactants needed for reverse micelle extraction. This can make the extraction process easier to operate and reduce chemical storage needed, potentially
AN
reducing the process costs.
The effects of feed aqueous phase pH on the forward extraction of amoxicillin
M
and erythromycin are shown in Fig. 1. The variation of antibiotic concentrations shown in Fig. 1 is typical for reverse micelle extraction of protein [36, 22]. In Figure 1(a), forward
ED
extraction efficiency of amoxicillin is higher at aqueous phase pH 4 and decreases when higher aqueous phase pH is used. Isoelectric point of amoxicillin is 4.7 [35]. Therefore,
PT
it will possess net positive charge in solution with pH less than 4.7 and on the other hand will possess net negative charge in solution with pH higher than 4.7. At aqueous phase
CE
pH 4, both amoxicillin and sophorolipids possess opposite net charges and thus are attracted to each other. When aqueous phase pH increased to 5 and 6, amoxicillin and
AC
sophorolipids are having same net charges and thus the amount of amoxicillin transferred to the reverse micellar phase gradually decreases. This indicates that electrostatic interactions between amoxicillin molecules and sophorolipids reverse micelles are significant during the forward extraction process. Similar effects of aqueous phase pH on the forward extraction of proteins are reported for tannase [37], lipase [16], and βglucosidase [19]. Fig. 1(a) also shows that forward extraction of amoxicillin is a rapid process. Amoxicillin concentration reaches a theoretical equilibrium value for each aqueous phase pH within 200 s of mixing time.
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Fig. 2 shows the application of Equation 12 for the forward extraction of amoxicillin and erythromycin. Combined mass transfer coefficients for amoxicillin are calculated from the gradients in Fig. 2(a). Individual mass transfer coefficients are obtained from Fig. 3(a) and the values are summarized in Table 1. When the feed aqueous phase pH is increased, less amoxicillin is solubilized into the organic phase and the transfer rate also decreases. This is due to the diminishing attractive electrostatic
T
interactions between amoxicillin molecules and sophorolipids reverse micelles at pH
IP
higher than pI of amoxicillin. The calculated values of korg,f A is larger than kaq,f A
CR
indicating that the rate limiting step for the forward extraction of amoxicillin is diffusion process in aqueous phase.
US
Fig. 1(b) shows the effect of feed aqueous phase pH on the forward extraction of erythromycin. The combined mass transfer coefficients for erythromycin are calculated
AN
from the gradients in Fig. 2(b). Individual mass transfer coefficients are obtained from Fig. 3(b) and the values are summarized in Table 2. Forward extraction of erythromycin
M
is also a rapid process as shown in Fig. 1(b). Theoretical equilibrium is reached within 100 s for all aqueous phase pH tested. The calculated values of combined mass transfer
ED
coefficients shows that the forward extraction rate decreases at higher aqueous phase pH. Erythromycin molecules net positive charge at solution pH below 8.6 [38], thus having
PT
stronger attractive electrostatic interactions with sophorolipids head groups, allowing more erythromycin molecules to be solubilized into the reverse micellar phase. When
CE
feed phase pH is further increased to above 8.6, erythromycin molecules mostly exist in non-polar form. Therefore, the attractive electrostatic interactions diminished and the
AC
mass transfer rates become lower. The calculated values of kaq,f A is significantly larger than korg,f A which indicates that the forward extraction of erythromycin is limited by the diffusion process in organic phase.
3.2
Backward Extraction The effects of stripping aqueous phase pH on the amount of amoxicillin and
erythromycin recovered to the aqueous phase are shown in Fig. 4. Fig. 4(a) shows that amoxicillin recovery is the lowest at stripping aqueous phase pH 5 because it is very close to the pI of amoxicillin, thus the attractive interactions are stronger compared to
ACCEPTED MANUSCRIPT 12
those at higher pH value. Amoxicillin recovery increases at pH 6 due to the increasing repulsive electrostatic interactions between amoxicillin molecules and sophorolipids. At aqueous phase pH 7, amoxicillin recovery decreases even though the repulsive electrostatic interactions should be higher than those at pH 6. This is due to the degradation of amoxicillin in neutral and alkaline medium [35]. Fig. 4(a) also shows that the theoretical equilibrium is achieved within 200 s of extraction time. This indicates that
T
the backward extraction can be a fast process.
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Fig. 5 shows the application of Equation 19 for the backward extraction of
CR
amoxicillin and erythromycin. Combined mass transfer coefficients for amoxicillin are calculated from the gradients in Fig. 5(a). Individual mass transfer coefficients are
US
obtained from Fig. 6(a) and the values are summarized in Table 3. The mass transfer rate increases when stripping aqueous phase pH increases from 5 to 6. This is due to the
AN
stronger repulsive electrostatic interactions between amoxicillin molecules and sophorolipids at solution pH higher than pI of amoxicillin. Data in Fig. 6(a) scatter far
M
away from a linear regression suggests that the mass transfer model may not be suitable to describe the backward extraction of amoxicillin at this pH range. This may due to the
ED
degradation of amoxicillin at stripping aqueous phase pH 7. Since one of the assumptions is that no chemical reaction is involved during the extraction process, the degradation of
PT
amoxicillin causes the calculated KbA for pH 7 to be not consistent with the calculated KbA for pH 5 and pH 6. Same inference is true for the mb values. Nevertheless, attempt
CE
on linear regression analysis shows that korg,b A is smaller than kaq,b A. Backward extraction of amoxicillin may be restricted by the diffusion process in organic phase.
AC
Fig. 4(b) shows the effect of stripping aqueous phase pH on the amount of erythromycin recovered to the aqueous phase. Backward extraction of erythromycin is a fast process as seen in Fig. 4(b) where the theoretical equilibrium is achieved within 200 s of extraction time. The increasing amount of erythromycin recovered when aqueous phase pH is increased from 8 to 10 is due to diminishing electrostatic interactions between erythromycin and sophorolipids, thus allowing more erythromycin molecules to be released into stripping phase [34]. Gaikaiwari et al. [37] also reported that maximum backward extraction efficiency is achieved at pH with minimum electrostatic interactions between biomolecules and reverse micelles.
ACCEPTED MANUSCRIPT 13
The combined mass transfer coefficients for erythromycin are calculated from the gradients in Fig. 5(b). Individual mass transfer coefficients are obtained from Fig. 6(b) and the values are summarized in Table 4. The mass transfer rate increases when stripping aqueous phase pH is increased from 8 to 10. The weakening of electrostatic interactions between erythromycin and sophorolipids allows faster release of erythromycin molecules from organic phase to aqueous phase. The calculated values of
IP
erythromycin is limited by the diffusion process in organic phase.
T
korg,b A is significantly lower than kaq,b A indicating that the backward extraction of
CR
The separation time needed for reverse micelle extraction of amoxicillin and erythromycin obtained in this study is very short compared to conventional solvent
US
extraction of antibiotics using butyl acetate which can take 35 h for separation process [39]. Reverse micelle extraction of penicillin G with chemical surfactants took more than
AN
15 min to achieve theoretical equilibrium [22]. Application of sophorolipids may have
3.3
M
enhanced the extraction rate of antibiotics.
Comparisons
ED
Amoxicillin and erythromycin belong to different class of antibiotics having different kind of properties. Erythromycin has more complex molecular structure and
PT
larger surface area compared to those of amoxicillin. These properties affect their behavior during the reverse micelle extraction process.
CE
The effects of aqueous phase pH on the mass transfer of amoxicillin and erythromycin during reverse micelle extraction were investigated because aqueous phase
AC
pH is the most dominant factor during the extraction process. Similar extraction behavior was observed when the aqueous phase pH was varied during the extraction of both antibiotics. This is due to the nature of both antibiotics to possess net negative charge at below their pI values, thus having strong attractive electrostatic interactions with the surfactant head groups. When aqueous phase pH is increased to higher than their pI values, the attractive electrostatic interactions weakened and promotes the release of antibiotics from reverse micellar phase into aqueous phase. Both antibiotics are very sensitive to the surrounding pH thus the whole extraction process should be conducted within their own pH range to avoid degradation of the antibiotics.
ACCEPTED MANUSCRIPT 14
The most significant difference between reverse micelle extraction of amoxicillin and erythromycin is the amount of antibiotics extracted during forward extraction and the amount of antibiotics recovered during backward extraction. Large mf and mb values obtained for the extraction of amoxicillin indicate that only small amount of amoxicillin is extracted or recovered under the extraction conditions used in this study. On the other hand, large amount of erythromycin can be extracted during forward extraction and
T
satisfactory amount of erythromycin can be recovered during backward extraction.
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Different sizes, molecular structures, and nature of the antibiotics should be the reason
CR
for this observation. The larger size and complex structure of erythromycin molecules may have allowed more interactions with the reverse micelles. The overall mb values in
US
this study are larger than mf values, showing that the backward extraction is more difficult compared to forward extraction. This is due to the strong attractive interactions
AN
between antibiotic molecules and sophorolipids head groups as well as stability of reverse micelles.
M
Theoretical equilibriums were achieved within 200 s for the reverse micelle extraction of both antibiotics. By further processing the mixture with fast phase
ED
separation equipment such as centrifuge, the antibiotic separation process can be completed in short time. The lower overall mass transfer coefficients of backward
PT
extraction compared to that of forward extraction been calculated shows that the backward extraction of antibiotics is more difficult than the forward extraction process.
CE
Reverse micelle extraction of erythromycin give a larger calculated overall mass transfer coefficients compared to amoxicillin, indicating that the extraction conditions examined
AC
in this study are more efficient for the separation of erythromycin than that of amoxicillin. Analysis on individual mass transfer coefficients shows that the reverse micelles extraction of erythromycin is mostly governed by the diffusion process in organic phase. On the other hand, the rate limiting step during forward extraction of amoxicillin is the diffusion process in aqueous phase. The rate limiting step during backward extraction of amoxicillin cannot be confirmed because the linear regression does not fit the data very well. Nevertheless, this observation suggests that there may be some differences on how amoxicillin and erythromycin molecules are solubilized into the organic phase or released into the stripping aqueous phase.
ACCEPTED MANUSCRIPT 15
4.0
Conclusion Mass transfer models to describe the reverse micelle extraction behavior of
antibiotics have been successfully developed and tested. The reverse micelle extraction is pH-dependent process. The forward and backward extractions of both antibiotics are found to be fast processes. Theoretical equilibriums were achieved within 200 s of
T
extraction time for all cases. The rate limiting step during forward extraction of
IP
amoxicillin is the diffusion process in aqueous phase but the rate limiting step during
CR
backward extraction of amoxicillin cannot be confirmed due to significant lack of fit. The diffusion process in organic phase is identified as the rate limiting step during both
US
forward and backward extractions of erythromycin. Reverse micelle extraction method in this study gives better performance for the separation of erythromycin compared to
AN
amoxicillin. This indicates that different properties possessed by different antibiotics have significant influence on their interactions with the reverse micelles, thus affecting
M
the outcomes of the reverse micelle extraction. Reverse micelle extraction has the potential to be applied in downstream processing of antibiotics. It can significantly save
ED
the cost and time of the separation of antibiotics. Furthermore, using biosurfactants such
CE
Acknowledgment
PT
as sophorolipids can ensure that the process is environmental friendly.
The authors thank the financial supports from Research Management Center
AC
(RMC), grant no. (Q.J130000.2546.17H02), UniversitiTeknologi Malaysia (UTM).
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Figure Captions:
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Fig. 1. Forward extraction of (a) amoxicillin and (b) erythromycin at different feed
CR
aqueous phase pH
Fig. 2. Forward extraction rates of (a) amoxicillin and (b) erythromycin at different feed
US
aqueous phase pH
Fig. 3. Mass transfer coefficients for forward extraction of (a) amoxicillin and (b)
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erythromycin
Fig. 4. Recovery of (a) amoxicillin and (b) erythromycin at different stripping aqueous
M
phase pH
Fig. 5. Backward extraction rates of (a) amoxicillin and (b) erythromycinat different
ED
stripping aqueous phase pH
Fig. 6. Mass transfer coefficients for backward extraction of (a) amoxicillin and (b)
CE
Table Legends:
PT
erythromycin
Table 1. Values of equilibrium partition coefficient and mass transfer coefficients for the
AC
forward extraction of amoxicillin at different feed aqueous phase pH Table 2. Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of erythromycin at different feed aqueous phase pH Table 3. Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of amoxicillin at different stripping aqueous phase pH Table 4. Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of erythromycin at different stripping aqueous phase pH
ACCEPTED MANUSCRIPT 21
Table 1: Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of amoxicillin at different feed aqueous phase pH KfA
4
2.077
5.240 × 10-8
5
2.636
3.919 × 10-8
6
9.000
1.748 × 10-8
kaq,f A
korg,f A
1.025 × 10-7
1.890 × 10-7
T
mf
AC
CE
PT
ED
M
AN
US
CR
IP
pH
ACCEPTED MANUSCRIPT 22
Table 2: Values of equilibrium partition coefficient and mass transfer coefficients for the forward extraction of erythromycin at different feed aqueous phase pH KfA
8
0.308
1.131 × 10-6
9
0.341
8.710 × 10-7
10
0.565
5.904 × 10-7
kaq,f A
korg,f A
1.386 × 10-5
3.459 × 10-7
T
mf
AC
CE
PT
ED
M
AN
US
CR
IP
pH
ACCEPTED MANUSCRIPT 23
Table 3: Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of amoxicillin at different stripping aqueous phase pH KbA
5
5.190
3.344 × 10-8
6
2.250
1.064 × 10-7
7
6.182
5.827 × 10-8
kaq,b A
korg,b A
3.210 × 10-7
2.139 × 10-7
T
mb
AC
CE
PT
ED
M
AN
US
CR
IP
pH
ACCEPTED MANUSCRIPT 24
Table 4: Values of equilibrium partition coefficient and mass transfer coefficients for the backward extraction of erythromycin at different stripping aqueous phase pH KbA
8
2.140
3.395 × 10-7
9
1.900
3.649 × 10-7
10
1.010
4.806 × 10-7
kaq,b A
korg,b A
1.318 × 10-6
7.625 × 10-7
T
mb
AC
CE
PT
ED
M
AN
US
CR
IP
pH
ACCEPTED MANUSCRIPT 25
Research Highlights: Eco-friendly sophorolipids biosurfactant was used in reverse micelle extraction of antibiotics Kinetic and mass transfers behavior were studied for reverse micelle extraction of
T
antibiotics
CR
IP
Effects of aqueous phase pH on the mass transfer was investigated for amoxicillin and erythromycin extraction
US
Reverse micelle extraction of antibiotics were completed within 200 s
AC
CE
PT
ED
M
AN
Forward and backward extraction methods were investigated
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6