Preparation of a novel thermo-sensitive copolymer forming recyclable aqueous two-phase systems and its application in bioconversion of Penicillin G

Separation and Purification Technology 75 (2010) 156–164

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Preparation of a novel thermo-sensitive copolymer forming recyclable aqueous two-phase systems and its application in bioconversion of Penicillin G Shu Miao, Jinpeng Chen, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Biochemical Engineering, East China University of Science & Technology, 130 Meilong Rd., Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 3 April 2010 Received in revised form 29 July 2010 Accepted 10 August 2010 Keywords: pH-sensitive polymer Thermo-sensitive polymer Aqueous two-phase systems Penicillin G 6-APA

a b s t r a c t Aqueous two-phase systems (ATPS) are potential industrial technology in bioseparation engineering and biocatalysis engineering areas. In industry, the main problem is that ATPS could not be recycled efficiently to result in a high cost and pollution of the environment. In this study, a new thermo-sensitive copolymer, poly(NIPA-co-BA) (abbreviated PNB ), has been synthesized by using N-isopropylacrylamide and N-butyl acrylate as monomers. It can form ATPS with another novel pH-sensitive copolymer poly(AA-coDMAEMA-co-BMA) (abbreviated PADB ). Based on the property of pH-thermo sensitivity, the copolymers could be recycled with over 97% recovery. Bioconversion of Penicillin G was carried out in the PNB –PADB ATPS. In the systems, optimized partition coefficients of substrate, Penicillin G, products 6-APA and phenylacetate acid were found to be: 3.87, 3.54 and 1.76, respectively. In the way, substrate and products are biased in top phase, while immobilized penicillin acylase is completely partitioned in bottom. Substrate, Penicillin G enters into bottom phase, and it is catalyzed into 6-APA and phenylacetate acid, then the products enter into top phase. Finally, inhibition of substrate and products is removed to result in improvement of products yield. The yield of product 6-APA could reach 95.4% and the value is higher 10% than in single aqueous phase. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The aqueous two-phase systems have been studied since 1950s. In the recent years, research interesting has been focused on the preparation and application of recycling polymers forming aqueous two-phase systems. Recovery of phase-forming polymer could be achieved by inducing precipitation with temperature, pH, ionic strength, and electric potential [1–5]. For example, when the temperature of polymer solution is raised above the lower critical solution temperature (LCST), the polymer is precipitated from solution. In this way, phase-forming polymer can be recycled at low cost. Thermo-sensitive ethylene oxide–propylene oxide copolymers (EO–PO) were firstly reported in early 1990s and they could form ATPS with Dextran. Johansson et al. [6,7] modified random copolymer of EO and PO with aliphatic C14 H29 groups and obtained an EOPO with hydrophobic groups (HM-EOPO). The modified HM-EOPO could form ATPS with mentioned EOPO polymer. Some preparations and applications of pH-sensitive and other

∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 1383-5866/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2010.08.003

type of thermo-sensitive polymers have been reported as well. Chen and Hoffman prepared copolymers of NIPAM and acrylic acid (AA) by grafting temperature-sensitive oligo-(NIPAM) chains onto backbone of a pH-sensitive polymer, and the polymer had responsive behavior to temperature or pH changes. Boyko et al. [8] synthesized a thermo-sensitive polymer, poly-N-vinylcaprolactam (PVCL) with LCST at 32 ◦ C. Yildiz et al. [9] synthesized a thermo-responsive random copolymer of N-isopropylacrylamide and N-hydroxymethyl acrylamide [poly(NIPAM-co-NHMAAm)]. Caykara et al. [10] also synthesized a new thermo-sensitive polymer poly(N-isopropylacrylamide-co-acrylamide) [P(NIPA-coAAm)]. As we can see, the N-isopropylacrylamide is a key monomer for thermo-sensitive polymer. Among the thermo-responsive polymers reported, poly(N-isopropylacrylamide) (PNIPA) homopolymer and its copolymers have been intensively investigated. In our lab, Cao and co-workers have synthesized a bunch of novel polymers to compose different ATPS used in different separation fields. Qin et al. [11] synthesized a novel pH-sensitive copolymer (PABC ), which was copolymerized, by using 2-(dimethylamino)ethyl methacrylate (DMAEMA), tertbutylmethacrylate (tBMA) and methyl methacrylate (MMA). The ATPS were formed by the PABC and PEG20000. The PABC could be recovered by adjusting isoelectric point (pI) to 8.4, with recovery of

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157

Fig. 1. Reaction of enzymatic hydrolysis of Pen G.

Fig. 2. Synthesis of pH-sensitive copolymer PADB .

Fig. 3. Synthesis of thermo-sensitive copolymer PNB .

95%. Kong et al. [12] synthesized the visible light-sensitive polymer PNBC which was copolymerized by using N-isopropylacrylamide (NIPA), n-butyl acrylate (BMA) and chlorophyllin sodium copper salt (CHL) as monomers. The ATPS was formed by the PNBC and dextran with 20,000 molecular mass. Over 95% of the PNBC could be recycled. Wang et al. [13] reported a novel light-sensitive copolymer (PNNC ) forming ATPSs with another pH-sensitive copolymer (PADB ). The copolymer (PNNC ) was synthesized by using NIPA, N-vinyl-2-pyrrolidone (NVP), CHL as monomers, and 2, 2 -azo-bisisobutyronitrile (AIBN) as initiator. The PNNC could be recycled through light radiation at 488 nm, and the PADB could be recycled at a mild pH range. Also Ning et al. [14] reported a novel pH-sensitive terpolymer PADB forming ATPS with another light-sensitive terpolymer PNBC . The PADB was synthesized by using acrylic acid (AA), DMAEMA, and BMA as monomers, and ammonium persulfate and sodium hydrogen sulfite as initiators. The PADB could be recycled at a mild pH (4.1) and PNBC could be recycled by light radiation at 488 nm.

Penicillin G acylase (PGA), an important industrial enzyme, catalyzes Penicillin G into 6-aminopenicillanic acid (6-APA). This enzyme has been applied in many industrial reactions such as hydrolysis of Penicillin G and cephalosporin G [15–17], synthesis of semisynthetic antibiotics [18]. Enzymatic hydrolysis of Penicillin G (Pen G) to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) as schematically represented in Fig. 1. This is a famous example of industrial biocatalysis. Cao’s group carried out research work on the bioconversion reaction of Penicillin G into 6-APA in ATPS. For example, Jin et al. [19] carried out bioconversion reaction of Penicillin G in The ATPS were composed by a light-sensitive polymer PNBC and Dextran 20000. The conversion ratio of Penicillin G was 82.6% (pH 7.8, 20 ◦ C), which was 20% higher that of the reaction in the single aqueous phase buffer. Then another ATPS was composed by PNBC and PADB , a light-sensitive polymer and a pH-sensitive polymer were used to bioconversion of Penicillin G [20], And the conversion ratio reached 85.3% (pH 7.8, 20 ◦ C). Low conversion yield was attributed to the reaction with low temperature due to polymer PNBC with low LCST. Here, we report a novel thermo-sensitive copolymer (PNB ) forming aqueous two-phase systems with a pH-sensitive copolymer (PADB ) synthesized by our laboratory. The copolymer (PNB ) was synthesized by using N-isopropylacrylamide (NIPA) and Nbutyl acrylate (BA) as monomers, and 2, 2 -Azo-bis-isobutyronitrile (AIBN) as initiator. The PNB could be recycled by temperature inducing at 33 ◦ C, and the PADB could be recycled at a mild pH range. It is believed that the thermo-pH-sensitive aqueous twophase systems will be very interesting in future biotechnology industry.

Table 1 The LCST of copolymers with different monomer ratio. A 1 2 3 4 5 6 7 8

NIPA

B

LCST (◦ C)

Acrylamide Butyl methacrylate Acrylic acid Methyl acrylate n-Butyl acrylate Acrylamide + butyl methacrylate Acrylamide + acrylic acid 1-Vinyl-2-pyrrolidone

28.0 30.0 28.5 29.3 34.1 28.0 28.3 28.0

Table 2 The LCST of copolymers with different monomer ratio.

1 2 3 4 5

M(NIPA):M(n-butyl acrylate) (w:w)

LCST (◦ C)

Recovery (%)

50:1 50:2 50:3 50:5 50:8

28.0 31.5 33.0 35.0 36.1

90.5 80.3 98.9 74.2 70

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Fig. 4. The structure analysis of PNB : the 1 H NMR spectrum of copolymer PNB .

2. Materials and methods 2.1. Chemicals N-(2-(Dimethylamino) ethyl)-methacrylamide (DMAEMA) was purchased from Wanduofu Chemical Co. (Zibo, Shandong Province, China). Acrylic acid (AA), n-butylmethacrylate (BMA), n-butyl acrylate (BA) and 2-2 -azoisobutyronitrile (AIBN) were obtained from Ling Feng Chemical Co. (Shanghai, China). N-Isopropylacrylamide (NIPA) was synthesized by reacting acryloyl chloride with isopropylamine and crystallized from hexane solution [21,22]. All chemicals were of analytical grade and were used without further purification. Penicillin G was purchased from North China Pharmaceutical Group Inc. Immobilized Penicillin G acylase (IMPA) (18 U/mg) was offered by LIVZON Pharmaceutical Group, Inc.

Fig. 6. The LCST of PNB . The five different concentrations of PNB samples were tested. During raise the temperature, record the process of their precipitation. Table 3 The recovery of PNB . No.

PNB (%)

1

2

3

4

5

98.6

98.6

98.0

97.9

97.5

The experiment was done when PNB was dissolved in pure water. The average recovery was 98.0% after five times. Table 4 The recoveries of PNB and PADB in ATPS. No.

Fig. 5. The phase diagram of PNB /PADB aqueous two-phase systems.

PNB (%) PADB (%)

1

2

3

4

5

6

96.9 96.5

97.1 97.0

97.4 97.5

98.1 97.4

97.3 97.1

97.7 97.3

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Fig. 7. Effect of salts on partition coefficient: (a) 6-APA; (b) PAA; (c) Penicillin G.

2.2. Preparation of P (AA-co-DMAEMA-co-BMA) (PADB ) The P (AA-co-DMAEMA-co-BMA) (PADB ) was synthesized as illustrated in Fig. 2. 10 ml acrylic acid, 8 ml DMAEMA and 1 ml n-butylmethacrylate were added into a conical flask containing 200 ml pure water at room temperature. Then the initiator (NH4 )2 S2 O8 and NaHSO3 were added into the solution by 1.3% (w/w). The reaction was carried out under the nitrogen atmosphere in a shaker for 24 h at 55 ◦ C. The product was filtrated and washed by absolute ethyl alcohol, then dried in the vacuum. 2.3. Preparation of P (NIPAM-co-BA) (PNB ) PNB was polymerized by using BA and NIPA as monomers, and AIBN as radical initiator in benzene solvent [23–25]. 10 g NIPA and 0.6 ml BA were added into a conical flask containing 40 ml benzene. The reaction was carried out under nitrogen atmosphere in a shaker for 24 h at 65 ◦ C. The schematic reaction formula was shown in Fig. 3. The product was dissolved in acetone and precipitated in hexane, and then the precipitate was dried in a vacuum drier at room temperature. 2.4. Characterization of PNB copolymer The PNB copolymer was characterized by using Bruker spectrospin DRX500 NMR equipment (Bruker Company, Switzerland). The

solvent was DMSO. In addition, the molecular weight of copolymer was measured by Umstatter viscometer measurement. 2.5. Phase-forming test and phase diagram Here, different concentrations of PADB were dissolved in 40 mM K2 HPO4 /KH2 PO4 buffer (pH 7.0), while the PNB is dissolved in purified water, to test possibility of forming ATPS. The mixtures were settled at room temperature for 4–5 h till the clear phase was observed. Take out the samples from top and bottom phases, and determine the concentrations of polymer PADB and PNB to draw the phase diagram. Then, the optimized ratio of PADB and PNB can be obtained to form the desired ATPS. 2.6. Recycling of copolymers 2.6.1. Recovery of PADB copolymer The pI of polymer could be measured according to Patrickios et al. [26]. The pH was adjusted to the pI of PADB . Then the precipitate will appear. We calculate the recovery as the ratio of the dry weight of precipitated polymer and that of the initial weight. 2.6.2. Recovery of PNB copolymer LCST of PNB is measured by raising the temperature from 22 to 40 ◦ C. The recovery of PNB was determined by weight of the polymer after thermo-precipitation.

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Fig. 8. The reactions of different concentrations of Penicillin G: (a) 3% Penicillin G with 5% PGA; (b) 5% Penicillin G with 5% PGA; (c) 8% Penicillin G with 5% PGA.

The precipitate drier.

was

filtrated

and

dried

in

the

vacuum

2.7. Partition of 6-APA, PAA and Penicillin G in ATPS In this experiment, 5% (w/w) 6-APA, 5% (w/w) PAA and 5% (w/w) Penicillin G were partitioned in PNB –PADB aqueous two-phase sys-

tems. The effect of salt species on partition was investigated by using KCL, NaCl, K2 SO4 , Na2 SO4 , (NH4 )2 SO4 , K3 PO4 , Na3 PO4 , KSCN, NaSCN and Na2 S2 O3 (as the species of the salts are so large, here we just pick some frequent salts from the lab to characterize the effects of the salts). The concentrations of all salts were from 10 to 80 mM with 10 mM step. Each salt was added into ATPS at above concentration. After ATPS reached equilibrium,

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Fig. 9. The reactions of different concentrations of PGA: (a) 1.6% PGA with 8% Penicillin G; (b) 5% PGA with 8% Penicillin G; (c) 8% PGA with 8% Penicillin G.

the samples were taken out from top and bottom phase by a syringe. The concentration of 6-APA, PAA and Penicillin G was determined by HPLC at 230 nm detection. Then partition coefficient was calculated by ratio of the concentrations of top and bottom phase.

2.8. Bioconversion reaction of Penicillin G in ATPS The reaction was carried out at 30 ◦ C in the shake flasks. Effect of different concentrations of Penicillin G and Penicillin acylase on 6APA yield were investigated. During reaction, the sample was taken out from top and bottom phase every several minutes to determine

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concentration of substrate and products until the reaction reached equilibrium.

2.9. HPLC analysis Here the HPLC measurement is used to determine the concentration of 6-APA, PAA and Penicillin G in the top phase and bottom phase. A reversed-phase column C18 (150 mm × 4.6 mm, 5 ␮m) from Phenomenex was used with the mobile phase consisted of acetonitrile 4 mM (A) and phosphate buffer (B) (VA :VB = 1:1), at flow rate of 1.0 ml/min, chromatograms were monitored by UV absorbance detection at 230 nm. The sample was diluted in 1.0 ml of mobile phase and then filtered through a 0.22 ␮m PVDF syringe filter and 20 ␮l sample was injected into the HPLC system.

3. Results and discussion 3.1. Synthesis of P (NIPAM-co-BA) (PNB ) We aim to synthesize a polymer that can form ATPS with PADB . The polymer should be recycled by temperature inducing. NIPA is a key thermo-sensitive monomer. The polymer was obtained successfully after many times tries. The results are given in Table 1. With different monomers, polymers with different LCST can be obtained. It has been seen that No. 5 polymer has LCST at 34.1 ◦ C. After the monomers are selected, it is need to find a high thermosensitive recovery of the polymer with change of monomer ratio. Table 2 shows the LCST and recoveries with the ratios of different monomers. Adjust the ratio of NIPA and n-butyl acrylate, the LCST and recovery of polymer would change as well. It can be seen that No. 3 polymer has the highest recovery (98.9%) when mole ratio of NIPA:n-butyl acrylate is 50:3. The LCST is determined by the ratio of NIPA. The LCST become higher when the ratio was low, but the high ratio of NIPA reduces the recovery of PNB . So we use this ratio of NIPA to n-butyl acrylate to ensure that the polymer has high recovery and desired LCST.

3.2. Characterization of copolymer PNB PNB can be recovered from its solution when raise the temperature to 33 ◦ C. The key monomer of PNB is NIPA, which is a hydrophilic monomer. Hydrophobic monomer, BA makes PNB hydrophobicity. The molecular weight can be calculated by using the Mark–Houwink formula. The molecular weight of copolymer PNB was 2.2 × 103 , measured by Ubbelohde Viscometer [27]. The 1 H NMR spectrum of copolymer PNB is shown in Fig. 4. Spectrum analysis of 1 H NMR is as follows: (1) the group –CH(CH3 )2 of NIPA has characteristic peaks at 4.01 (–CH) and 1.13 (–CH3 ). And there are peaks at 3.88 and 1.13 in this spectrum. Its ratio could be deduced by the area of peaks; (2) characteristic peak of BA: the –(CH2 )3 –CH3 of N-butyl acrylate should be at 0.9, 1.3, 1.6 and 4.1. At this spectrum, there are peaks in 1.29, 1.67 and 4.29. It inferred that the PNB contains monomer BA. And the ratio of BA could be calculated by the area of peak. According to the area of the group peaks and the amount of hydrogen atoms, the molar ratio of each monomer contained in the PNB copolymer could be estimated. The monomer ratio of this copolymer is 4.97:0.42 (NIPA:NB), and the ratio is similar to the feed ratio of this copolymer, 5:0.3 (NIPA:NB). By spectral analysis, it is deduced that the copolymer PNB contains the two monomers in proportion.

3.3. Possibility to form aqueous two-phase systems and phase diagram Different concentration of PNB and PADB were mixed for investigation of forming ATPS. It can be seen that ATPS could be formed when same volume 5% (w/w) PADB and 5% (w/w) PNB were mixed. Fig. 5 is the phase diagram of PNB –PADB . The phase separation is driven by repulsion between the polymer PNB and PADB . The PNB is mainly enriched in the top while the PADB is mainly in bottom phase. The region above the bimodal curve is the two-phase region. M point represents the total composition of the copolymers in the ATPS. T and B on the bimodal curve describe the concentration of polymers in the top and bottom phases. The ratio of phases could be calculated according to length ratio of line MB and MT on tie line TMB. For the optimized volume ratio for bioconversion reaction is 3:1 (VTop :VBottom ). In the volume ratio, an optimized reaction yield could be obtained. 3.4. Recovery of PNB /PADB 3.4.1. Recovery of PADB by pH pH of the solution was adjusted to pI by HCl and precipitate could be separated by centrifugation and was used to form ATPS again. The pI of PADB is 4. As known to all, the pI of 6-APA is 4.1. It brings us a problem that when adjusting pH, they will precipitate together. So during the process of the experiment we use organic solution such as toluene to extract 6-APA.Also, in order to overcome this problem, our team is working on it and several ways are putting into practice. Up till now, we have tried to change the ratio of the monomer of PADB to change its pI to 3.9 which is different from the pI of 6-APA; to use new separation methods, ultrafiltration. 3.4.2. Recovery of PNB by temperature The LCST of PNB is measured by raising the temperature from 22 to 40 ◦ C, which is shown in Fig. 6. The LCST of PNB is nearly 33 ◦ C. The reason for such behavior could be attributed due to a dual hydrophilic/hydrophobic nature of thermo-sensitive copolymers. When above their LCST, thermo-sensitive polymers become unsoluble because of hydrogen bonds interrupt in polymer. Table 3 indicates that recoveries of PNB copolymer were recycled five times. Table 4 indicates the recovery of the two polymers in ATPS. The average recoveries of PNB and PADB were 97.4 and 97.1%, respectively. After using 20 times, their recoveries still keep in a very high level. 3.5. Partition of 6-APA, PAA and Penicillin G in ATPS The partition coefficients of 6-APA, PA and Penicillin G in ATPS were determined in this experiment. Generally, different inorganic salts can affect the partition of biomolecules in ATPS in some extent. Here ten inorganic salts including KCL, NaCl, K2 SO4 , Na2 SO4 , (NH4 )2 SO4 , K3 PO4 , Na3 PO4 , KSCN, NaSCN and Na2 S2 O3 were used to improve the partition coefficient. In the presence of 20 mM KCl, the maximum coefficients were obtained as 3.54, 1.76 and 3.87 for 6-APA, PA and Penicillin G, respectively. The detail was shown in Fig. 7. As the results shown, it is a positive effect because 6-APA, PA and Penicillin G can enrich in the top phase with larger volume. PGA is completely partitioned in the bottom phase. In the ATPS, 6-APA, PA and Penicillin G have negative charge. PADB also has negative charge and repulse these molecules with same charge into the top phase. These inorganic salts mainly enhance the partition coefficients of 6-APA, PA and Penicillin G by affecting the interface potential. Different species of ions have different partition behavior in the two phases. Electronic neutrality

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has to be maintained in each phase. The electrochemical driving force in partitioning could be explained by the formation of an electrostatic potential difference over the interface. This potential difference is created by the different species of the ions in the two phases (Donan effect). The electrostatic potential difference can affect the partitioning of biomolecules with charge in the phase system. 3.6. Bioconversion reaction of Penicillin G The hydrolytic reactions in the ATPS were carried out with various conditions. During the process, the partition coefficients of 6-APA, PA and Penicillin G were examined. When initial concentrations of Penicillin G were at 3%, 5% and 8% (w/w) and concentration of PGA was fixed at 5% (w/w), the reaction equilibrium time was 400, 460 and 480 min, respectively. As shown in Fig. 8, the more Penicillin G added in the reaction, the longer time it takes to reach equilibrium. In Fig. 9, when the concentrations of PGA were at 1.6, 5 and 8% (w/w) and concentration of Penicillin G was fixed 8% (w/w), the equilibrium time was 600, 480 and 460 min, respectively. In all data, the concentrations of Penicillin G decrease sharply in the first 50–100 min, while concentration of products, 6-APA and PAA increase with decrease of Penicillin G. It could be seen that the partition coefficient changes within a very small range. As mentioned, the interface potential is the key factor for substances with charges. With reaction going on, the components of reaction system become more complicated. The concentrations of products become higher in the system. 6-APA and phenylacetate with negative charges cause slightly change of interface potential difference. Penicillin acylase can affect the reaction equilibrium time, and also affects the yield of conversion of the Penicillin G in a small extent. At lower concentration of the substrate, yield of 6-APA and PAA could reach 95.4 and 94.8%, respectively. The conversion of Penicillin G is 95% or so. The bioconversion in ATPS was compared with the reaction in single aqueous system, and the yield of 6-APA in ATPS is higher 10% than that in single aqueous phase. Because 6-APA and PADB have near pI, we have to avoid the pI of 6-APA. PADB was synthesized with pI at 4.5–4.8. After reaction finished, the recovery of PNB and PADB was also carried according to aforementioned method. 4. Conclusions In this study, a novel thermo-sensitive polymer PNB has been synthesized with N-isopropylacrylamide and n-butyl acrylate as monomers by random polymerization. It can be recovered at 33 ◦ C or higher. ATPS can be formed by PNB and pH-sensitive polymer PADB . PADB is also a recoverable polymer that can be precipitated when the pH of the solution is 4.5. Both recoveries of the polymers are over 97%. In the presence of 20 mM KCl, The partition coefficient of 6-APA, PA and Penicillin G can reach 3.54, 1.76 and 3.87, respectively. When the reaction reaches equilibrium, the ratio of convention of Penicillin G is 95% and the yield of 6-APA is 95.4%. Since the maximum temperature of PGA bioconversion reaction is 37 ◦ C, while our experiment can only carried out at 30 ◦ C because of the limitation of the thermo-sensitive polymer. Though the process of separating PADB and 6-APA yet is not quite satisfied, we are now looking for some new methods and have made some progress. The ATPS could be easily recycled at low cost. There is no any environment problem. It can be used for bioseparation engineering and biocatalysis engineering in biotechnology industry. The result of the reaction is affected by many factors. In this

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