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Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase
Biochemical Engineering Journal 79 (2013) 136–143
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase夽 Jingjing Liu, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineerng, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 14 July 2013 Accepted 21 July 2013 Available online 30 July 2013 Keywords: Aqueous two phase Cellulose Cellulase Immobilized enzymes Biodegradation pH-response copolymer
a b s t r a c t Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated PADB3.8 ) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated PMDB ). In the systems, cellulase was immobilized on pH-response copolymer PMDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH4 )2 SO4 . Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cellulose, one of the most abundant and widespread renewable natural resources on the earth is being developed as a source of fuels for the future [1]. Hydrolysis of cellulose into reducing sugar by complex enzyme systems has been proved to be a promising process to obtain various products [2]. Cellulose saccharification is catalyzed by complex cellulase systems including cellobiohydrolases (EC 3.2.1.91), endoglucanases (EC 3.2.1.4), and -glucosidases (EC 3.2.1.21) [3]. However, the difficulty in the recovery of cellulase leads to high cost for the enzymatic hydrolysis of cellulosic biomass. Covalently immobilizing enzyme on reversibly soluble-insoluble polymer hopefully solves the problem of high cost in bioconversion processes. The polymers can be recovered easily by changing conditions, such as pH, temperature and light [4–6]. Especially, acrylate polymers are attractive polymers for enzymes immobilization. Acrylate polymers are stable to microorganism and acidic and
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.07.009
basic environments. The polymers are relatively easy to be modified for immobilization reaction [4,7]. Enzymatic degradation of polysaccharides to monosaccharide is a key step in production of chemicals from cellulosic biomass [8]. Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. Enzymatic hydrolysis efficiency of cellulose is usually decreased due to product inhibition [9]. Since Albertsson discovered aqueous two-phase systems (ATPS) in 1950s [10], the systems have shown an attractive prospect in bioseparation [11–13] and bioconversion areas [14,15]. In recent years, more and more bioconversion reactions were carried out in aqueous two-phase systems to remove the inhibition effect. Unfortunately, recovery of phase-forming copolymers could not be achieved to result in high cost and environmental pollution. In our laboratory, Cao’s group has done much work on the exploration of novel phase-forming copolymers and their applications in bioseparation [16,17] and bioconversion areas [18,19]. Miao et al. reported that a thermo-response copolymer poly (NIPAco-BA) (PNB ) could form ATPS with the pH-response copolymer poly (AA-co-DMAEMA-co-BMA) (PADB4.1 ). Bioconversion of Penicillin G was carried out in the PNB /PADB4.1 ATPS. The yield of product 6aminopenicillanic acid (6-APA) could reach 95.4% finally [18]. Li and Cao reported bioconversion of cephalosporin G to 7-Amino3-deacetoxy cephalospornic acid (7-ADCA) in a novel pH-thermo recyclable aqueous two-phase systems, and the conversion ratio of
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cephalosporin G could reach 98.57% at initial concentration of 12% (w/v), while the yield of 7-ADCA was 97.56% in PNB /PADB2.8 ATPS [19]. Recently, novel pH–pH recyclable ATPS composed by two pHresponse copolymers PADB3.8 and PMDB have been developed in our laboratory. In this study, the systems were applied to carry out degradation of cellulose. Cellulase was immobilized on pHresponse copolymer PMDB . The novel PADB3.8 /PMDB ATPS showed a promising application in carrying out the phase transfer bioconversion. The novel ATPS achieved the recyclability of cellulase with high recovery and it also improved the saccharification yield due to reducing the product inhibition. This is the first report of degradation of cellulose in pH–pH recyclable ATPS.
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adjusting pH to the isoelectric point (pH 3.8), and the recovery of PADB3.8 could be calculated by the percentage of the dry weight to that of the initial dry weight. 2.5. Reducing sugar assays The concentration of reducing sugar was measured by Dinitrosalicylic Acid (DNS) method. 3.0 ml DNS reagent was added into a test tube containing 2.0 ml diluted samples. The tube was heated in boiling water for 10 min and then cooled to the room temperature. The samples were diluted to 25 ml and the absorbance was measured by spectrophotometer at 540 nm [20]. 2.6. Partition of reducing sugar in ATPS
2. Materials and methods 2.1. Materials ␣-Methacrylic acid (MAA), acrylic acid (AA), butyl methacrylate (BMA), ammonium persulfate (APS) and sodium hydrogen sulfite (NaHSO3 ) were purchased from Ling Feng Chemical Co. (Shanghai, China). 2-dimethylamino ethyl methacrylate (DMAEMA) was obtained from Wanduofu Chemical Co. (Zibo, Shandong Province, China). All other chemicals were of analytical grade and were used without further purification. Celluclast 1.5 L FG was obtained from Novozymes (Denmark). Microcrystalline cellulose was purchased from YuanJu Biotechnol Co. (Shanghai, China). 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide hydrochloride (EDC) was obtained from sigma (Shanghai, China). 2.2. Preparation of two copolymers Copolymer PMDB was synthesized by others in our lab [4]. Copolymer PADB3.8 was synthesized by using AA, DMAEMA, BMA as monomers, and ammonium persulfate and sodium hydrogen sulfate as initiators [17]. 5.0 ml AA, 3.5 ml DMAEMA and 0.5 ml BMA were added into a conical flask containing 120 ml of deionized water. Then initiators were added into the solution by 1.3% (w/w). The polymerization reaction was carried out under nitrogen protection for 24 h at 55 ◦ C. The product was collected by centrifugation and washed three times by absolute ethyl alcohol, then dried in the vacuum.
In this experiment, the reaction product was reducing sugar. The effects of various salts and chaotropic agents on product partition was investigated by using KCl, KBr, K2 SO4 , KSCN, NaCl, NaBr, Na2 SO4 , NaSCN, NH4 Cl, (NH4)2 SO4 , MgSO4 , guanidine hydrochloride and urea. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. PADB3.8 /PMDB ATPS with different concentration of salts were put into water bath at 50 ◦ C for 10 min, then the partition coefficient of reducing sugar with different concentration of salts in ATPS was measured. Samples taken from top and bottom phases were determined by spectrophotometer at 540 nm. The partition coefficient was expressed as glucose concentration ratio of top phase to bottom phase. Temperature is an important factor in partition. Considering the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS reached a maximum value with 40 mM (NH4 )2 SO4 at 50 ◦ C, three relevant kinds of salts ((NH4 )2 SO4 , Na2 SO4 and NH4 Cl) containing NH4 + and SO4 2− at the concentration of 40 mM were selected to investigate the effect of temperature on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. PADB3.8 /PMDB ATPS with different kinds of salts ((NH4 )2 SO4 , Na2 SO4 and NH4 Cl) were put into water bath at 30 ◦ C, 40 ◦ C, 50 ◦ C and 60 ◦ C for 10 min, respectively. Then, the partition coefficient of reducing sugar with different kinds of salts was measured. Samples taken from top and bottom phases were determined by spectrophotometer at 540 nm. 2.7. Biodegradation reaction of cellulose in ATPS
2.3. Cellulase immobilization One gram of PMDB was dissolved in 50 ml NaOH (0.5 M) in a beaker with constant stirring, and pH of the solution was adjusted to 6.0 by dropwise adding 3 M HCl. Then, 300 mg EDC and 1.2 ml Cellucalst 1.5 L (150 mg protein) were added to the copolymer solution with stirring. pH of the mixture was adjusted to 3.5 by using 3 M acetic acid after being stirred gently at room temperature for 4 h. The precipitate was collected by centrifugation (8000 rpm for 15 min at 4 ◦ C) and the product was washed three times using distilled water with the same pH value (pH = 3.5). The immobilized enzyme was stored at 4 ◦ C. 2.4. Recycle of copolymer One gram copolymer PADB3.8 was dissolved in 20 ml NaOH solution (150 mM). 0.1 M HCl was added to the solution until precipitation weight appeared the maximum value. Then pH of the solution was considered to be the isoelectric point (pI) of copolymer PADB3.8 . One gram copolymer PADB3.8 was dissolved in 20 ml NaOH solution (150 mM). Copolymer PADB3.8 could be precipitated by
Reducing sugar was produced by using cellulase as a catalyst and microcrystalline cellulose as substrate in PADB3.8 /PMDB ATPS. In the study, effects of different reaction temperatures on saccharification yield were investigated. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 10 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 0.25 g PADB3.8 and 0.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.5% (w/v). Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker with 150 rpm at 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 60 ◦ C, respectively. Samples taken from top and bottom phases every 6 h and biodegradation reaction continued until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by the following equation [21]: Yieldofsaccharification(%) = Reducingsugar(g) × 0.89/Carbohydrates(g) × 100%
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Effects of different initial substrate concentrations (0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v)) on cellulose biodegradation reaction in PADB3.8 /PMDB ATPS were investigated. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 10 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 0.25 g PADB3.8 and 0.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v), respectively. Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker at150 rpm (50 ◦ C). Samples taken from top and bottom phases every 6 h and biodegradation reaction continued until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. Finally, reactions in PADB3.8 /PMDB ATPS and in the single aqueous phase were performed. Cellulase was immobilized on copolymer PMDB before both reactions. Copolymers were dissolved in 150 mM NaOH solution and pH values of both ATPS and single aqueous phase were adjusted to 5.5 by dropwise adding 150 mM NaOH solution. PADB3.8 /PMDB ATPS was composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. The concentration of (NH4)2 SO4 was 40 mM. Single aqueous phase was composed by 2.5% (w/v) PMDB with immobilized cellulase. In the experiment, both reactions were carried out in a shaker at 150 rpm (50 ◦ C). Substrate concentration was 0.5% (w/v). When the reaction finished, the tube was centrifuged at 4 ◦ C, 8000 × g for 20 min to accelerate the phase separation. The sample was taken out from top and bottom phases every 6 h to determine the concentration of product until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. Reusability of immobilized cellulase in recyclable PADB3.8 /PMDB ATPS was also investigated by hydrolysis of microcrystalline cellulose. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 50 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 1.25 g PADB3.8 and 1.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.5% (w/v). Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker at150 rpm (50 ◦ C). After 72 h reaction, the sample of first circle was taken out from top and bottom phase after phase separation. Since pH of PABD3.8 /PMDB ATPS used for cellulose biodegradation reaction was 5.5, copolymers and immobilized cellulase were completely dissolved in aqueous solution at pH 5.5. Therefore, insoluble substrate was removed by centrifuged at 4◦ Cfor 20 min. Copolymer PMDB with immobilized cellulase had the maximum precipitate value at pH 3.5 according to Liang’s publication [4]. Copolymer PADB3.8 had a maximum precipitate value at pH 3.8, while the precipitate ratio of copolymer PMDB with immobilized cellulase could also reach 80% [4]. Thus, the reusability of immobilized cellulase in PADB3.8 /PMDB ATPS was investigated by collecting the mixture of precipitates (including PADB3.8 and PMDB ) at pH 3.8 and pH 3.5. Firstly, pH of solution was adjusted to the isoelectric point of PADB3.8 (pH = 3.8) and precipitate was collected by centrifugation. Then, pH of supernatant was adjusted to the isoelectric point of PMDB with immobilized cellulase (pH = 3.5) and precipitate was collected by centrifugation. Two precipitates were mixed together,
Table 1 The phase-forming test of copolymers PADB and copolymers PMDB a .
1 2 3 4 5 6 7
Copolymer PADB with different monomer ratiob
Copolymer PMDB c with immobilized enzymed
Formation of ATPS
23:8:1 23:7:1 23:6:1 23:5:1 23:4:1 23:3:1 23:2:1
PMDB PMDB PMDB PMDB PMDB PMDB PMDB
NO YES NO NO NO NO NO
a The mixture solution consists 2.5% (w/v) PADB and 2.5% (w/v) PMDB . Solution pH was 5.5. b Monomer ratio of copolymer PADB indicates M(acrylic acid): M(DMAEMA): M(butyl methacrylate) (w:w). c Monomer ratio of copolymer PMDB indicates M(␣-Methacrylic): M(DMAEMA): M(butyl methacrylate) (w:w) was 19:1:1. d Immobilization conditions: reaction time is 4 h; pH is 6.0; EDC amount is 300 mg/g PMDB .
and dissolved again in 50 ml NaOH solution (150 mM). Then fresh substrate was added into the tube (0.5% (w/v)). The same operation was repeated five times. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. 3. Results and discussion 3.1. Synthesis of copolymer PADB3.8 A novel copolymer PADB3.8 was developed in our laboratory. It was synthesized by modified method of copolymer PADB4.1 according to Ning’s publication [17]. The monomer molar ratio of AA, DMAEMA and BMA of PADB4.1 was changed. PADB3.8 was named because its isoelectric point (pI) was 3.8. Copolymer PMDB was synthesized by others in our laboratory according to Liang’s publication [4]. As shown in Table 1, PADB with different monomer molar ratio of AA, DMAEMA, and BMA was synthesized to test the possibility of forming ATPS with pH-response copolymer PMDB . Copolymers PMDB and PADB with different monomer molar ratio were dissolved in 150 mM NaOH solution. pH of the mixture solution was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The mixture solution consisted 2.5% (w/v) PMDB and 2.5% (w/v) PADB . The systems were investigated whether it could form ATPS after centrifugation. PADB is a pH-response copolymer, which has acid group and alkaline group. Monomer AA is an acid monomer with negative charge, while monomer DMAEMA is an alkaline monomer with positive charge. These two monomers were responsible for the pH sensitivity of the copolymer. Monomer BMA was used for regulating the hydrophobicity of the copolymer. The interactions between PMDB and PADB with different monomer molar ratio such as Van der Waals force and hydrophobic interaction were also different. Therefore, PADB with different monomer molar ratio have different intermolecular attraction with PMDB and other components. In addition, PADB with different monomer molar ratio also had different molecular weight, which had a significant effect on forming ATPS. It is necessary to prepare a copolymer with the most suitable monomer molar ratio (the special monomer molar ratio of AA/DMAEMA/BMA) to form ATPS with PMDB . It could be seen from results that only the monomer molar ratio of AA/DMAEMA/BMA of PADB3.8 was 23:7:1 could form ATPS with copolymer PMDB . The phase separation phenomenon could be attributed to the interaction between the segments of PMDB and PADB3.8 at the special monomers molar ratio. It could be seen from Table 1, copolymer PADB with other monomer molar ratio could not form ATPS
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the aqueous solution. The maximum precipitate of PADB3.8 occurred at pH 3.8, which was considered as its isoelectric point. Fig. 1(b) shows the recovery of novel copolymer PADB3.8 after recycled five times. Each recovery was the average value of three parallel experimental points. The recovery of copolymer PADB3.8 kept more than 96.7% after the five cycles with relation to initial amount. The values of recovery were quite stable. This indicated that the copolymer PADB3.8 could still kept 50% after 40 recycles. 3.3. Partition of reducing sugar in PADB3.8 /PMDB ATPS
Fig. 1. (a) The recovery of copolymer PADB3.8 at different pH value. 0.1 M HCl solution was added to 5% (w/v) PADB3.8 solution until precipitation appeared. Precipitate at varied pH value was collected and the recovery of PADB3.8 could be calculated by the percentage of the dry weight to that of the initial weight. (b) The recycling recovery of copolymer PADB3.8 . PADB3.8 was precipitated by adjusting pH to 3.8, and collected by centrifugation at 8000 rpm. The precipitate was dried to a constant weight and dissolved again. The recovery of PADB3.8 was calculated as the percentage of the dry weight to that of the initial weight.
with PMDB . One possible explanation was that only the interactions between PMDB molecules and PADB3.8 molecules in aqueous solution were repulsive in character. PADB3.8 and PMDB molecules preferred to be surrounded by their own kind instead of being mixed. Therefore, the result of mixing solutions of PMDB and PADB3.8 was incompatibility. As a result, PADB3.8 /PMDB system could form ATPS when the systems reached equilibrium [10]. In this experiment, the novel recyclable PADB3.8 /PMDB ATPS were used. 3.2. Recycle of copolymer PADB3.8 The recoveries of copolymer PADB4.1 and PMDB have been studied previously. The average recoveries of PADB4.1 and PMDB were 97.1% and 95.4%, respectively [18,22]. It was mainly focused on the recovery of novel copolymer PADB3.8 in this study. Fig. 1(a) shows the precipitation ratio of novel copolymer PADB3.8 in pH range of 3.2–4.8. It could be seen from results that increasing pH to above 4.8 or decreasing pH to below 3.2, PADB3.8 was completely soluble in
Cellulase was immobilized on copolymer PMDB , and the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS was investigated in this experiment. Reducing sugar was evenly partitioned between top phase and bottom phase without any neutral salts. Different salt types with varied concentration had different effect on partitioning of reducing sugar. The effect of salts and chaotropic agents (KCl, KBr, K2 SO4 , KSCN, NaCl, NaBr, Na2 SO4 , NaSCN, NH4 Cl, (NH4)2 SO4 , MgSO4 , guanidine hydrochloride and urea) on partition coefficient of reducing sugar was investigated from 10 mM to 90 mM concentrations with 10 mM step at 50 ◦ C. Each salt was added into ATPS at aforementioned concentration. The samples were taken out from top and bottom phases after ATPS reached equilibrium. Then, the concentration of glucose in the top phase and in the bottom phase was measured, respectively. Partition coefficient could be calculated by glucose concentration ratio of top phase to bottom phase. The results were shown in Fig. 2(a). As the results shown, reducing sugar was enriched in top phase and the partition coefficient was 2.45, in presence of 40 mM (NH4)2 SO4 . From the Fig. 2(a), it could be seen that K2 SO4 , Na2 SO4 , MgSO4 and NH4 Cl did not show good distribution function of reducing sugar in PADB3.8 /PMDB ATPS, and only (NH4)2 SO4 in certain concentration (40 mM) had effect on partitioning reducing sugar in PADB3.8 /PMDB ATPS. Partition between two phases depends on many factors since interactions between the partitioned substance and the components of each phase are a complex phenomenon involving hydrogen bonds, charge interaction, Van der Waals forces, hydrophobic interactions, and steric effects [10]. Different species of salt ions in ATPS may lead to unevenly distribution of all the components in the system. The partition of reducing sugar in this study depends on the molecular weight and chemical properties of copolymer PADB3.8 and copolymer PMDB , and the size and chemical properties of the partitioned molecules or particles including reducing sugar and salt ions. Theoretically, the substance partitioning into a phase could be increased by decreasing the molecular weight of the polymer enriched in that phase, or by increasing the molecular weight of the polymer constituting the opposite phase [10]. In the experiment, cellulase could not be immobilized on copolymer PMDB and aqueous two-phase systems could not form if we increasing or decreasing the molecular weight of either copolymer. In this ATPS, copolymer PMDB with immobilized cellulase was one amphiphilic copolymer enriched in bottom phase, while copolymer PADB3.8 with acid monomer (AA) and alkaline monomer (DMAEMA) was the other amphiphilic copolymer enriched in top phase. One of possible explanation for the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS was that the existence of (NH4)2 SO4 in certain concentration (40 mM) could change the intermolecular attraction including Van der Waals force, hydrophobic interaction between product and copolymers, which made reducing sugar distribute unevenly between two phases. The intermolecular attraction between reducing sugar and copolymer PADB3.8 might be bigger than that between reducing sugar and copolymer PMDB , and then produced forces to drive reducing sugar into the top phase. Interactions including hydrophobic interactions, hydrogen bonds, charge effect determine the partitioning of product.
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Fig. 2. (a) Effects of salts and chaotropic agents on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. The partition coefficient was measured at 50 ◦ C. (b) Effects of temperature on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The partition coefficient of reducing sugar with 40 mM Na2 SO4 , NH4 Cl and (NH4 )2 SO4 in PADB3.8 /PMDB ATPS was measured at different temperature, respectively.
Microcrystalline cellulose as substrate in the study was insoluble, so it was completely partitioned in the bottom phase with immobilized cellulase in PADB3.8 /PMDB ATPS due to larger density of particles than that of water phase or large surface of the particles. In contrast to the soluble molecules, insoluble particulates partition between two phases and the interface, and with increasing size between on phase and the interface. The excess of substrate would be distributed in interface between two phases. Similar observation was previously reported [10]. The effect of temperature on partition coefficient of reducing sugar was shown in Fig. 2(b). The partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS showed a slight increase with the increasing of temperature. Partition coefficient of reducing sugar with 40 mM (NH4)2 SO4 was 2.09 at 30 ◦ C, and it increased to 2.61 when the temperature increased to 60 ◦ C. One possible explanation
was that the increasing temperature could increase the thermal motion of partitioning substance and the interactions between partitioning substance and copolymers. Therefore, the partition coefficient of reducing sugar had a slight increase when the temperature increased. 3.4. Biodegradation reaction of microcrystalline cellulose 3.4.1. Effects of reaction temperature on the cellulose biodegradation The hydrolysis of cellulose had been carried out in PNB /PMDB ATPS in previous work according to Liu’s publication [22]. Liang reported the optimum reaction temperature for cellulose biodegradation was 50 ◦ C [4]. However, the experiment in PNB /PMDB ATPS could only be carried out under 30 ◦ C because of the limitation
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when the temperature was above 50 ◦ C. Therefore, saccharification yield reached the maximum value at 50 ◦ C and 50 ◦ C was chosen as the optimum reaction temperature for cellulose biodegradation in the study. In summary, the novel PADB3.8 /PMDB ATPS showed a promising application in biodegradation of cellulose because it solved the problem of temperature limitation of thermo- response copolymer PNB in PNB /PMDB ATPS.
Fig. 3. Effects of reaction temperature on bioconversion of microcrystalline cellulose. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The biodegradation reaction was carried out in a shaker with 150 rpm at 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 60 ◦ C, respectively. The concentration of cellulose was 0.5% (w/v). The sample was taken out from top and bottom phases every 6 h to determine the concentration of reducing sugar until 72 h.
of thermo- response copolymer PNB . Therefore, yield of saccharification was limited and results were not quite satisfied. In this study, temperature limitation during biodegradation of cellulose in ATPS was solved. Novel pH–pH recyclable ATPS consisted of two pHresponse copolymers PADB3.8 and PMDB could be applied at higher temperature. Cellulase was immobilized on pH-response copolymer PMDB . The pH stability and thermo stability of free cellulase were improved by immobilizing cellulase on copolymer PMDB . The optimum pH and temperature of the pH-response copolymer PMDB with immobilized cellulase was also reported by Liang [4]. The effect of reaction temperature on the bioconversion in PADB3.8 /PMDB ATPS was firstly investigated. Fig. 3 shows the effect of reaction temperature on biodegradation of cellulose from 30 ◦ C to 60 ◦ C. From the results, it could be seen that saccharification yield increased sharply until 54 h at different reaction temperature, while after 54 h, saccharification yield maintained stable. With the reaction temperature increased from 30 ◦ C to 50 ◦ C, saccharification yield increased from 40.71% to 70.57% after 54 h. It could be concluded that the novel PADB3.8 /PMDB ATPS have obvious advantages over PNB /PMDB ATPS. Therefore, saccharification yield was improved greatly in PADB3.8 /PMDB ATPS after remove temperature limitation. When the reaction temperature went on increasing from 50 ◦ C to 60 ◦ C, saccharification yield decreased from 70.57% to 60.85% after 54 h. Inactivation of cellulase at such high temperature may lead to the decrease in saccharification yield. The thermal stability of PMDB with immobilized cellulase was reported in Liang’s publication [4]. It demonstrated that the absolute activity of immobilized cellulase decreased more than 30% when the temperature increased from 50 ◦ C to 60 ◦ C. Similar observations were also reported in Ince’s publication [23] and Dincer’s publication [24]. Ince et al. [23] investigated the effect of temperature on the catalytic activity of immobilized cellulase. It suggested that the optimum temperature for cross-linked cellulase was 50 ◦ C. The relative activity of cellulase increased obviously from 15 ◦ C to 50 ◦ C and decreased a lot from 50 ◦ C to 75 ◦ C. Dincer and Telefoncu [24] also demonstrated that the remaining activity of immobilized cellulase decreased a lot
3.4.2. Effects of initial substrate concentration on the cellulose biodegradation The effect of initial substrate concentration on cellulose biodegradation reaction was shown in Fig. 4(a). For the PADB3.8 /PMDB ATPS, the initial concentration of microcrystalline cellulose was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The reaction temperature was chosen 50 ◦ C. It could be seen that the concentration of substrate had obvious effect on saccharification yield. From the results, saccharification yield could reach 70.57% when the substrate concentration was 0.5% (w/v). The value was 5.72% higher than the reaction with 0.25% (w/v) substrate and 19.79% higher than the reaction with 1% (w/v) substrate. An increase of substrate concentration from 0.5% (w/v) to 1% (w/v) resulted in relatively obvious decrease of cellulose biodegradation. The marked decrease in saccharification yield at high substrate concentration (1% (w/v)) resulted in an optimum substrate concentration (0.5% (w/v)). It could be seen from Fig. 4(b) that the concentration of reducing sugar significantly increased from 3.92% (w/v) to 5.65% (w/v) linearly when the substrate concentration increased from 0.5% (w/v) to 1% (w/v). The concentration of the released reducing sugar could even reach 6.05% (w/v) at high substrate concentration (1% (w/v) substrate) after 72 h. Such high concentration of reducing sugar may also lead to the decrease in reaction rate because of product inhibition. Andrew et al. [25] used the model to explore various potential rate-limiting phenomena during cellulose biodegradation, such as substrate accessibility, product inhibition and the molecular weight of the cellulose substrate. Although cellulose was insoluble in this experiment, substrate concentration had obvious effect on cellulose biodegradation. Too low initial substrate concentration might not be enough for cellulase biocatalytic reaction, while too high initial substrate concentration in the reaction medium might limit the yield of saccharification due to decreasing mobility of insoluble substrate in the aqueous phase of the reaction mixture or increasing the viscosity of the reaction mixture which had an adverse effect on the mobility of the reactants and the release of products. Similar observations were also previously reported [26–28]. 3.4.3. Cellulose biodegradation in PADB3.8 /PMDB ATPS and in the single aqueous phase Cellulase was immobilized on copolymer PMDB before reaction. Biodegradation reactions in PADB3.8 /PMDB ATPS with immobilized cellulase were carried out in the experiment. ATPS was composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. Biodegradation with immobilized cellulase in the single aqueous phase was conducted as a control experiment. Single aqueous phase used for biodegradation reaction was composed by 2.5% (w/v) PMDB which immobilized cellulase on. Both reactions were carried out in a shaker at 150 rpm (50 ◦ C). Initial substrate concentration was 0.5% (w/v). Fig. 5 shows the comparison of two reactions carried out in PADB3.8 /PMDB ATPS and in the single aqueous phase. In PADB3.8 /PMDB ATPS, insoluble substrate and immobilized cellulase were biased to bottom phase, and product was partitioned to top phase in presence of 40 mM (NH4)2 SO4 . Microcrystalline cellulose was catalyzed into reducing sugar, and products entered into the top phase. In this way, inhibition of products was removed and
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Fig. 5. Bioconversion of cellulose in ATPS and in single aqueous phase. Cellulase was immobilized on copolymer PMDB . The initial concentration of substrate was 0.5% (w/v). The reaction of biodegradation of microcrystalline cellulose was carried out in PADB3.8 /PMDB ATPS and single aqueous phase in a shaker at 50 ◦ C. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS.
Fig. 4. (a) Effects of initial substrate concentration on bioconversion of cellulose. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The biodegradation reaction was carried out in a shaker with 150 rpm at 50 ◦ C. The sample was taken out from top and bottom phases every 6 h to determine the concentration of reducing sugar until 72 h. (b) The concentration of reducing sugar with different initial substrate concentration. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The biodegradation reaction was carried out in a shaker with 150 rpm at 50 ◦ C.
saccharification yield was improved in ATPS compared with that in the single aqueous phase. The saccharification yield and reducing sugar concentration in ATPS were 74.34% and 4.13% (w/v) after 72 h, respectively. While the values in the single aqueous phase were 65.47% and 3.64% (w/v), respectively. The accumulated products in single aqueous phase during batch reaction inhibited hydrolytic rate. The similar observations were also emphasized by Andric et al. [9] and Gan et al. [29]. The saccharification yield in PADB3.8 /PMDB
ATPS was improved greatly compared with the result of previous work in PNB /PMDB ATPS [22]. Although the increase of saccharification yield was limited because of low partition coefficient of product, these novel recyclable ATPS certainly had advantages in industrial production because the new systems solved the problem of product inhibition during biocatalytic reaction and saccharification yield in PADB3.8 /PMDB ATPS was improved compared with the reaction in the single aqueous phase. The final saccharification yield still had a large space for improvement. We are now still looking for other salts to improve the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS. If the partition coefficient could be increased much more, saccharification yield should also be increased much higher. Furthermore, many other bioconversion reactions could also be carried out in this novel recyclable ATPS not limited to cellulose biodegradation. Product inhibition could also be solved and products yield would be increased compared with that in the single aqueous phase.
Fig. 6. Reusability of immobilized enzyme in recyclable PADB3.8 /PMDB ATPS. The reaction was carried out in a shaker with 0.5% (w/v) substrate at 50 ◦ C.
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3.4.4. Reusability of immobilized cellulase in PADB3.8 /PMDB ATPS After biodegradation reaction, both copolymers and immobilized cellulase could be recovered with high recovery and low cost by aforementioned method. The saccharification yield could still reach 66.15% after immobilized cellulase was recycled five times in recyclable PADB3.8 /PMDB ATPS (Fig. 6). Possible factors causing the decrease of saccharification yield included the loss of immobilized cellulase during polymer recovery process and unavoidable enzyme deactivation during centrifuge separation and biodegradation reaction. 4. Conclusions In this study, novel recyclable PADB3.8 /PMDB ATPS with watersoluble immobilized cellulase were used to biodegradation of cellulose. Cellulase was immobilized on PMDB , with over 80% reserved enzyme activity. The recycling recoveries of both copolymers were over 95%. A significant advantage is that phase-forming copolymers and cellulase could be recycled at a very low cost for many times. On the other hand, phase transfer bioconversion of cellulose has been carried out in PADB3.8 /PMDB ATPS. The saccharification yield in ATPS could reach 70.57% after 54 h, 9.3% higher than that in the single aqueous phase. The saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS. The yield of saccharification in PADB3.8 /PMDB ATPS had been improved greatly compared with the result of previous work in PNB /PMDB ATPS. The novel PADB3.8 /PMDB ATPS could also be applied to other phase transfer bioconversion. References [1] D. Ciolacu, S. Gorgieva, D. Tampu, V. Kokol, Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose, Cellulose 18 (2011) 1527–1541. [2] R. Smith, Biodegradable Polymers for Industrial Applications, Woodhead Publishing, London, 2005. [3] N. Ortega, M.D. Busto, M. Perez-Mateos, Kinetics of cellulose saccharification by trichoderma reeseri cellulases, International Biodeterioration and Biodegradation 47 (2001) 7–14. [4] W.J. Liang, X.J. Cao, Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization, Bioresource Technology 116 (2012) 140–146. [5] Y. Zhang, J.L. Xu, D. Li, Z.H. Yuan, Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100, Biocatalysis and Biotransformation 28 (2010) 313–319. [6] J.Q. Zhou, Immobilization of cellulase on a reversibly soluble-insoluble support: properties and application, Journal of Agricultural and Food Chemistry 58 (2010) 6741–6746. [7] M.A. Abd El-Ghaffar, K.S. Atia, M.S. Hashem, Synthesis, Characterization of binary copolymers of methyl methacrylate with glycidyl methacrylate and 2hydroxy ethyl methacrylate as carriers for cellulase, Journal of Applied Polymer Science 117 (2010) 629–638. [8] Y.J. Han, H.Z. Chen, Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase, Bioresource Technology 102 (2011) 4787–4792.
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[9] P. Andric, A.S. Meyer, P.A. Jensen, K. Dam-Johansen, Reactor design for minimizing product inhibition during enzymatic lignocelluloses hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes, Biotechnology Advances 28 (2010) 308–324. [10] P.A. Albertsson, Partition of Cell Particles and Macromolecules, Third ed., John Wiley & Sons, Inc., Canada, 1986, pp. 1–110. [11] O. Aguilar, M. Rito-Palomares, Aqueous two-phase systems strategies for the recovery and characterization of biological products from plants, Journal of the Science of Food and Agriculture 90 (2010) 1385–1392. [12] R. Dembczynski, W. Bialas, T. Jankowski, Recycling of phase components during lysozyme extraction from hen egg white in the EO50 PO50 /K2 HPO4 aqueous twophase system, Biochemical Engineering Journal 51 (2010) 24–31. [13] F.C. de Oliveira, J.S.R. Coimbra, L.H.M. da Silva, E.E.G. Rojas, M.C.H. da Silva, Ovomucoid partitioning in aqueous two-phase systems, Biochemical Engineering Journal 47 (2009) 55–60. [14] M. Canales, C. Ballesteros, J.A. Moreno-Cid, A.M. Espinosa, M. Villar, J. de la Fuente, Extractive bioconversion to produce the Aedes albopictus akirin in an aqueous two-phase system supporting Pichia pastoris growth and protein secretion, Biochemical Engineering Journal 46 (2009) 105–114. [15] X. Li, Z. Lian, B. Dong, Y. Xu, Q. Yong, S.Y. Yu, Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG6000/sodium citrate aqueous two-phase system, Chemical Engineering Journal 28 (2011) 1897–1901. [16] W. Wang, J.F. Wan, B. Ning, J.N. Xia, X.J. Cao, Preparation of a novel lightresponse copolymer and its application in recycling aqueous two-phase systems, Journal of Chromatography A 1205 (2008) 171–176. [17] B. Ning, J.F. Wan, X.J. Cao, Preparation and recycling of aqueous two-phase systems with pH-response amphiphilic terpolymer PADB , Biotechnology Progress 25 (2009) 820–824. [18] S. Miao, J.P. Chen, X.J. Cao, Preparation of a novel thermo-response copolymer forming recyclable aqueous two-phase system and its application in bioconversion of penicillin G, Separation and Purification Technology 75 (2010) 156–164. [19] H. Li, X.J. Cao, Bioconversion of cephalosporin-G to 7-ADCA in a pH-thermo sensitive recycling aqueous two-phase systems, Process Biochemistry 46 (2011) 1753–1758. [20] T.K. Ghose, Measurement of cellulase activities, Pure and Applied Chemistry 59 (1987) 257–268. [21] J. Szczodrak, The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol, Biotechnology and Bioengineering 32 (1988) 771–776. [22] J.J. Liu, X.J. Cao, Biodegradation of cellulose in novel recyclable aqueous twophase systems with water-soluble immobilized cellulase, Process Biochemistry 47 (2012) 1998–2004. [23] A. Ince, G. Bayramoglu, B. Karagoz, B. Altintas, N. Bicak, M.Y. Arica, A method for fabrication of polyaniline coated polymer microspheres and its application for cellulase immobilization, Chemical Engineering Journal 189-190 (2012) 404–412. [24] A. Dincer, A. Telefoncu, Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads, Journal of Molecular Catalysis B: Enzymatic 45 (2007) 10–14. [25] J.G. Andrew, J.S. Jonathan, J.L. James, A Mechanistic Model for Enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition, Biotechnology and Bioengineering 109 (2012) 676–685. [26] A.F. Abdel-Fattah, M.A. Abdel-Naby, Pretreatment and enzymic saccharification of water hyacinth cellulose, Carbohydrate Polymers 87 (2012) 2109–2113. [27] M. Chen, J. Zhao, L.M. Xia, Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars, Carbohydrate Polymers 71 (2008) 411–415. [28] Y. Zheng, Z.Y. Pan, R.H. Zhang, D.H. Wang, Enzymatic saccharification of dilute acid pretreated saline crops for fermentable sugar production, Applied Energy 86 (2009) 2459–2465. [29] Q. Gan, S.J. Allen, G. Taylor, Kinetic dynamics in heterogeneous enzymatic hydrolysis of cellulose: an overview, an experimental study and mathematical modeling, Process Biochemistry 38 (2003) 1003–1018.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Biodegradation of microcrystalline cellulose in pH–pH recyclable aqueous two-phase systems with water-soluble immobilized cellulase夽 Jingjing Liu, Xuejun Cao ∗ State Key Laboratory of Bioreactor Engineering, Department of Bioengineerng, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 14 July 2013 Accepted 21 July 2013 Available online 30 July 2013 Keywords: Aqueous two phase Cellulose Cellulase Immobilized enzymes Biodegradation pH-response copolymer
a b s t r a c t Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. In addition, the difficulty in the recovery of cellulase also leads to high cost for the enzymatic hydrolysis of cellulose. In this study, enzymatic degradation of cellulose was carried out in pH–pH recyclable aqueous two-phase systems (ATPS) composed by copolymers poly (AA-co-DMAEMA-co-BMA) (abbreviated PADB3.8 ) and poly (MAA-co-DMAEMA-co-BMA) (abbreviated PMDB ). In the systems, cellulase was immobilized on pH-response copolymer PMDB by using 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC) as cross-linker. Optimized partition coefficient of product in the systems was 2.45, in the presence of 40 mM (NH4 )2 SO4 . Insoluble substrate and immobilized enzyme were biased to bottom phase, while the product was partitioned to top phase. Microcrystalline cellulose was hydrolyzed into reducing sugar, and the product entered into top phase. The yield of saccharification in ATPS could reach 70.57% at the initial substrate concentration of 0.5% (w/v), and the value was 9.3% higher than that in the single aqueous phase. Saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Cellulose, one of the most abundant and widespread renewable natural resources on the earth is being developed as a source of fuels for the future [1]. Hydrolysis of cellulose into reducing sugar by complex enzyme systems has been proved to be a promising process to obtain various products [2]. Cellulose saccharification is catalyzed by complex cellulase systems including cellobiohydrolases (EC 3.2.1.91), endoglucanases (EC 3.2.1.4), and -glucosidases (EC 3.2.1.21) [3]. However, the difficulty in the recovery of cellulase leads to high cost for the enzymatic hydrolysis of cellulosic biomass. Covalently immobilizing enzyme on reversibly soluble-insoluble polymer hopefully solves the problem of high cost in bioconversion processes. The polymers can be recovered easily by changing conditions, such as pH, temperature and light [4–6]. Especially, acrylate polymers are attractive polymers for enzymes immobilization. Acrylate polymers are stable to microorganism and acidic and
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel.: +86 21 64252695; fax: +86 21 64252695. E-mail address: [email protected] (X. Cao). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.07.009
basic environments. The polymers are relatively easy to be modified for immobilization reaction [4,7]. Enzymatic degradation of polysaccharides to monosaccharide is a key step in production of chemicals from cellulosic biomass [8]. Product inhibition is a barrier for enzymatic conversion of cellulose into reducing sugar in single aqueous phase. Enzymatic hydrolysis efficiency of cellulose is usually decreased due to product inhibition [9]. Since Albertsson discovered aqueous two-phase systems (ATPS) in 1950s [10], the systems have shown an attractive prospect in bioseparation [11–13] and bioconversion areas [14,15]. In recent years, more and more bioconversion reactions were carried out in aqueous two-phase systems to remove the inhibition effect. Unfortunately, recovery of phase-forming copolymers could not be achieved to result in high cost and environmental pollution. In our laboratory, Cao’s group has done much work on the exploration of novel phase-forming copolymers and their applications in bioseparation [16,17] and bioconversion areas [18,19]. Miao et al. reported that a thermo-response copolymer poly (NIPAco-BA) (PNB ) could form ATPS with the pH-response copolymer poly (AA-co-DMAEMA-co-BMA) (PADB4.1 ). Bioconversion of Penicillin G was carried out in the PNB /PADB4.1 ATPS. The yield of product 6aminopenicillanic acid (6-APA) could reach 95.4% finally [18]. Li and Cao reported bioconversion of cephalosporin G to 7-Amino3-deacetoxy cephalospornic acid (7-ADCA) in a novel pH-thermo recyclable aqueous two-phase systems, and the conversion ratio of
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cephalosporin G could reach 98.57% at initial concentration of 12% (w/v), while the yield of 7-ADCA was 97.56% in PNB /PADB2.8 ATPS [19]. Recently, novel pH–pH recyclable ATPS composed by two pHresponse copolymers PADB3.8 and PMDB have been developed in our laboratory. In this study, the systems were applied to carry out degradation of cellulose. Cellulase was immobilized on pHresponse copolymer PMDB . The novel PADB3.8 /PMDB ATPS showed a promising application in carrying out the phase transfer bioconversion. The novel ATPS achieved the recyclability of cellulase with high recovery and it also improved the saccharification yield due to reducing the product inhibition. This is the first report of degradation of cellulose in pH–pH recyclable ATPS.
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adjusting pH to the isoelectric point (pH 3.8), and the recovery of PADB3.8 could be calculated by the percentage of the dry weight to that of the initial dry weight. 2.5. Reducing sugar assays The concentration of reducing sugar was measured by Dinitrosalicylic Acid (DNS) method. 3.0 ml DNS reagent was added into a test tube containing 2.0 ml diluted samples. The tube was heated in boiling water for 10 min and then cooled to the room temperature. The samples were diluted to 25 ml and the absorbance was measured by spectrophotometer at 540 nm [20]. 2.6. Partition of reducing sugar in ATPS
2. Materials and methods 2.1. Materials ␣-Methacrylic acid (MAA), acrylic acid (AA), butyl methacrylate (BMA), ammonium persulfate (APS) and sodium hydrogen sulfite (NaHSO3 ) were purchased from Ling Feng Chemical Co. (Shanghai, China). 2-dimethylamino ethyl methacrylate (DMAEMA) was obtained from Wanduofu Chemical Co. (Zibo, Shandong Province, China). All other chemicals were of analytical grade and were used without further purification. Celluclast 1.5 L FG was obtained from Novozymes (Denmark). Microcrystalline cellulose was purchased from YuanJu Biotechnol Co. (Shanghai, China). 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide hydrochloride (EDC) was obtained from sigma (Shanghai, China). 2.2. Preparation of two copolymers Copolymer PMDB was synthesized by others in our lab [4]. Copolymer PADB3.8 was synthesized by using AA, DMAEMA, BMA as monomers, and ammonium persulfate and sodium hydrogen sulfate as initiators [17]. 5.0 ml AA, 3.5 ml DMAEMA and 0.5 ml BMA were added into a conical flask containing 120 ml of deionized water. Then initiators were added into the solution by 1.3% (w/w). The polymerization reaction was carried out under nitrogen protection for 24 h at 55 ◦ C. The product was collected by centrifugation and washed three times by absolute ethyl alcohol, then dried in the vacuum.
In this experiment, the reaction product was reducing sugar. The effects of various salts and chaotropic agents on product partition was investigated by using KCl, KBr, K2 SO4 , KSCN, NaCl, NaBr, Na2 SO4 , NaSCN, NH4 Cl, (NH4)2 SO4 , MgSO4 , guanidine hydrochloride and urea. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. PADB3.8 /PMDB ATPS with different concentration of salts were put into water bath at 50 ◦ C for 10 min, then the partition coefficient of reducing sugar with different concentration of salts in ATPS was measured. Samples taken from top and bottom phases were determined by spectrophotometer at 540 nm. The partition coefficient was expressed as glucose concentration ratio of top phase to bottom phase. Temperature is an important factor in partition. Considering the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS reached a maximum value with 40 mM (NH4 )2 SO4 at 50 ◦ C, three relevant kinds of salts ((NH4 )2 SO4 , Na2 SO4 and NH4 Cl) containing NH4 + and SO4 2− at the concentration of 40 mM were selected to investigate the effect of temperature on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. PADB3.8 /PMDB ATPS with different kinds of salts ((NH4 )2 SO4 , Na2 SO4 and NH4 Cl) were put into water bath at 30 ◦ C, 40 ◦ C, 50 ◦ C and 60 ◦ C for 10 min, respectively. Then, the partition coefficient of reducing sugar with different kinds of salts was measured. Samples taken from top and bottom phases were determined by spectrophotometer at 540 nm. 2.7. Biodegradation reaction of cellulose in ATPS
2.3. Cellulase immobilization One gram of PMDB was dissolved in 50 ml NaOH (0.5 M) in a beaker with constant stirring, and pH of the solution was adjusted to 6.0 by dropwise adding 3 M HCl. Then, 300 mg EDC and 1.2 ml Cellucalst 1.5 L (150 mg protein) were added to the copolymer solution with stirring. pH of the mixture was adjusted to 3.5 by using 3 M acetic acid after being stirred gently at room temperature for 4 h. The precipitate was collected by centrifugation (8000 rpm for 15 min at 4 ◦ C) and the product was washed three times using distilled water with the same pH value (pH = 3.5). The immobilized enzyme was stored at 4 ◦ C. 2.4. Recycle of copolymer One gram copolymer PADB3.8 was dissolved in 20 ml NaOH solution (150 mM). 0.1 M HCl was added to the solution until precipitation weight appeared the maximum value. Then pH of the solution was considered to be the isoelectric point (pI) of copolymer PADB3.8 . One gram copolymer PADB3.8 was dissolved in 20 ml NaOH solution (150 mM). Copolymer PADB3.8 could be precipitated by
Reducing sugar was produced by using cellulase as a catalyst and microcrystalline cellulose as substrate in PADB3.8 /PMDB ATPS. In the study, effects of different reaction temperatures on saccharification yield were investigated. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 10 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 0.25 g PADB3.8 and 0.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.5% (w/v). Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker with 150 rpm at 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 60 ◦ C, respectively. Samples taken from top and bottom phases every 6 h and biodegradation reaction continued until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by the following equation [21]: Yieldofsaccharification(%) = Reducingsugar(g) × 0.89/Carbohydrates(g) × 100%
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Effects of different initial substrate concentrations (0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v)) on cellulose biodegradation reaction in PADB3.8 /PMDB ATPS were investigated. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 10 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 0.25 g PADB3.8 and 0.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v), 1% (w/v), respectively. Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker at150 rpm (50 ◦ C). Samples taken from top and bottom phases every 6 h and biodegradation reaction continued until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. Finally, reactions in PADB3.8 /PMDB ATPS and in the single aqueous phase were performed. Cellulase was immobilized on copolymer PMDB before both reactions. Copolymers were dissolved in 150 mM NaOH solution and pH values of both ATPS and single aqueous phase were adjusted to 5.5 by dropwise adding 150 mM NaOH solution. PADB3.8 /PMDB ATPS was composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. The concentration of (NH4)2 SO4 was 40 mM. Single aqueous phase was composed by 2.5% (w/v) PMDB with immobilized cellulase. In the experiment, both reactions were carried out in a shaker at 150 rpm (50 ◦ C). Substrate concentration was 0.5% (w/v). When the reaction finished, the tube was centrifuged at 4 ◦ C, 8000 × g for 20 min to accelerate the phase separation. The sample was taken out from top and bottom phases every 6 h to determine the concentration of product until 72 h. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. Reusability of immobilized cellulase in recyclable PADB3.8 /PMDB ATPS was also investigated by hydrolysis of microcrystalline cellulose. Cellulase was immobilized on copolymer PMDB before biodegradation reaction. The reaction was carried out in 50 ml ATPS composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. 1.25 g PADB3.8 and 1.25 g PMDB with immobilized cellulase were dissolved in 150 mM NaOH solution, and pH was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The initial concentration of substrate was 0.5% (w/v). Then, the desired amount of (NH4)2 SO4 was added into the PADB3.8 /PMDB ATPS to adjust the partition coefficient of product, and the concentration of (NH4)2 SO4 was 40 mM. The reaction was carried out in a shaker at150 rpm (50 ◦ C). After 72 h reaction, the sample of first circle was taken out from top and bottom phase after phase separation. Since pH of PABD3.8 /PMDB ATPS used for cellulose biodegradation reaction was 5.5, copolymers and immobilized cellulase were completely dissolved in aqueous solution at pH 5.5. Therefore, insoluble substrate was removed by centrifuged at 4◦ Cfor 20 min. Copolymer PMDB with immobilized cellulase had the maximum precipitate value at pH 3.5 according to Liang’s publication [4]. Copolymer PADB3.8 had a maximum precipitate value at pH 3.8, while the precipitate ratio of copolymer PMDB with immobilized cellulase could also reach 80% [4]. Thus, the reusability of immobilized cellulase in PADB3.8 /PMDB ATPS was investigated by collecting the mixture of precipitates (including PADB3.8 and PMDB ) at pH 3.8 and pH 3.5. Firstly, pH of solution was adjusted to the isoelectric point of PADB3.8 (pH = 3.8) and precipitate was collected by centrifugation. Then, pH of supernatant was adjusted to the isoelectric point of PMDB with immobilized cellulase (pH = 3.5) and precipitate was collected by centrifugation. Two precipitates were mixed together,
Table 1 The phase-forming test of copolymers PADB and copolymers PMDB a .
1 2 3 4 5 6 7
Copolymer PADB with different monomer ratiob
Copolymer PMDB c with immobilized enzymed
Formation of ATPS
23:8:1 23:7:1 23:6:1 23:5:1 23:4:1 23:3:1 23:2:1
PMDB PMDB PMDB PMDB PMDB PMDB PMDB
NO YES NO NO NO NO NO
a The mixture solution consists 2.5% (w/v) PADB and 2.5% (w/v) PMDB . Solution pH was 5.5. b Monomer ratio of copolymer PADB indicates M(acrylic acid): M(DMAEMA): M(butyl methacrylate) (w:w). c Monomer ratio of copolymer PMDB indicates M(␣-Methacrylic): M(DMAEMA): M(butyl methacrylate) (w:w) was 19:1:1. d Immobilization conditions: reaction time is 4 h; pH is 6.0; EDC amount is 300 mg/g PMDB .
and dissolved again in 50 ml NaOH solution (150 mM). Then fresh substrate was added into the tube (0.5% (w/v)). The same operation was repeated five times. The concentration of reducing sugar was measured by DNS method, and the yield of saccharifiaction was calculated by aforementioned equation. 3. Results and discussion 3.1. Synthesis of copolymer PADB3.8 A novel copolymer PADB3.8 was developed in our laboratory. It was synthesized by modified method of copolymer PADB4.1 according to Ning’s publication [17]. The monomer molar ratio of AA, DMAEMA and BMA of PADB4.1 was changed. PADB3.8 was named because its isoelectric point (pI) was 3.8. Copolymer PMDB was synthesized by others in our laboratory according to Liang’s publication [4]. As shown in Table 1, PADB with different monomer molar ratio of AA, DMAEMA, and BMA was synthesized to test the possibility of forming ATPS with pH-response copolymer PMDB . Copolymers PMDB and PADB with different monomer molar ratio were dissolved in 150 mM NaOH solution. pH of the mixture solution was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The mixture solution consisted 2.5% (w/v) PMDB and 2.5% (w/v) PADB . The systems were investigated whether it could form ATPS after centrifugation. PADB is a pH-response copolymer, which has acid group and alkaline group. Monomer AA is an acid monomer with negative charge, while monomer DMAEMA is an alkaline monomer with positive charge. These two monomers were responsible for the pH sensitivity of the copolymer. Monomer BMA was used for regulating the hydrophobicity of the copolymer. The interactions between PMDB and PADB with different monomer molar ratio such as Van der Waals force and hydrophobic interaction were also different. Therefore, PADB with different monomer molar ratio have different intermolecular attraction with PMDB and other components. In addition, PADB with different monomer molar ratio also had different molecular weight, which had a significant effect on forming ATPS. It is necessary to prepare a copolymer with the most suitable monomer molar ratio (the special monomer molar ratio of AA/DMAEMA/BMA) to form ATPS with PMDB . It could be seen from results that only the monomer molar ratio of AA/DMAEMA/BMA of PADB3.8 was 23:7:1 could form ATPS with copolymer PMDB . The phase separation phenomenon could be attributed to the interaction between the segments of PMDB and PADB3.8 at the special monomers molar ratio. It could be seen from Table 1, copolymer PADB with other monomer molar ratio could not form ATPS
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the aqueous solution. The maximum precipitate of PADB3.8 occurred at pH 3.8, which was considered as its isoelectric point. Fig. 1(b) shows the recovery of novel copolymer PADB3.8 after recycled five times. Each recovery was the average value of three parallel experimental points. The recovery of copolymer PADB3.8 kept more than 96.7% after the five cycles with relation to initial amount. The values of recovery were quite stable. This indicated that the copolymer PADB3.8 could still kept 50% after 40 recycles. 3.3. Partition of reducing sugar in PADB3.8 /PMDB ATPS
Fig. 1. (a) The recovery of copolymer PADB3.8 at different pH value. 0.1 M HCl solution was added to 5% (w/v) PADB3.8 solution until precipitation appeared. Precipitate at varied pH value was collected and the recovery of PADB3.8 could be calculated by the percentage of the dry weight to that of the initial weight. (b) The recycling recovery of copolymer PADB3.8 . PADB3.8 was precipitated by adjusting pH to 3.8, and collected by centrifugation at 8000 rpm. The precipitate was dried to a constant weight and dissolved again. The recovery of PADB3.8 was calculated as the percentage of the dry weight to that of the initial weight.
with PMDB . One possible explanation was that only the interactions between PMDB molecules and PADB3.8 molecules in aqueous solution were repulsive in character. PADB3.8 and PMDB molecules preferred to be surrounded by their own kind instead of being mixed. Therefore, the result of mixing solutions of PMDB and PADB3.8 was incompatibility. As a result, PADB3.8 /PMDB system could form ATPS when the systems reached equilibrium [10]. In this experiment, the novel recyclable PADB3.8 /PMDB ATPS were used. 3.2. Recycle of copolymer PADB3.8 The recoveries of copolymer PADB4.1 and PMDB have been studied previously. The average recoveries of PADB4.1 and PMDB were 97.1% and 95.4%, respectively [18,22]. It was mainly focused on the recovery of novel copolymer PADB3.8 in this study. Fig. 1(a) shows the precipitation ratio of novel copolymer PADB3.8 in pH range of 3.2–4.8. It could be seen from results that increasing pH to above 4.8 or decreasing pH to below 3.2, PADB3.8 was completely soluble in
Cellulase was immobilized on copolymer PMDB , and the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS was investigated in this experiment. Reducing sugar was evenly partitioned between top phase and bottom phase without any neutral salts. Different salt types with varied concentration had different effect on partitioning of reducing sugar. The effect of salts and chaotropic agents (KCl, KBr, K2 SO4 , KSCN, NaCl, NaBr, Na2 SO4 , NaSCN, NH4 Cl, (NH4)2 SO4 , MgSO4 , guanidine hydrochloride and urea) on partition coefficient of reducing sugar was investigated from 10 mM to 90 mM concentrations with 10 mM step at 50 ◦ C. Each salt was added into ATPS at aforementioned concentration. The samples were taken out from top and bottom phases after ATPS reached equilibrium. Then, the concentration of glucose in the top phase and in the bottom phase was measured, respectively. Partition coefficient could be calculated by glucose concentration ratio of top phase to bottom phase. The results were shown in Fig. 2(a). As the results shown, reducing sugar was enriched in top phase and the partition coefficient was 2.45, in presence of 40 mM (NH4)2 SO4 . From the Fig. 2(a), it could be seen that K2 SO4 , Na2 SO4 , MgSO4 and NH4 Cl did not show good distribution function of reducing sugar in PADB3.8 /PMDB ATPS, and only (NH4)2 SO4 in certain concentration (40 mM) had effect on partitioning reducing sugar in PADB3.8 /PMDB ATPS. Partition between two phases depends on many factors since interactions between the partitioned substance and the components of each phase are a complex phenomenon involving hydrogen bonds, charge interaction, Van der Waals forces, hydrophobic interactions, and steric effects [10]. Different species of salt ions in ATPS may lead to unevenly distribution of all the components in the system. The partition of reducing sugar in this study depends on the molecular weight and chemical properties of copolymer PADB3.8 and copolymer PMDB , and the size and chemical properties of the partitioned molecules or particles including reducing sugar and salt ions. Theoretically, the substance partitioning into a phase could be increased by decreasing the molecular weight of the polymer enriched in that phase, or by increasing the molecular weight of the polymer constituting the opposite phase [10]. In the experiment, cellulase could not be immobilized on copolymer PMDB and aqueous two-phase systems could not form if we increasing or decreasing the molecular weight of either copolymer. In this ATPS, copolymer PMDB with immobilized cellulase was one amphiphilic copolymer enriched in bottom phase, while copolymer PADB3.8 with acid monomer (AA) and alkaline monomer (DMAEMA) was the other amphiphilic copolymer enriched in top phase. One of possible explanation for the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS was that the existence of (NH4)2 SO4 in certain concentration (40 mM) could change the intermolecular attraction including Van der Waals force, hydrophobic interaction between product and copolymers, which made reducing sugar distribute unevenly between two phases. The intermolecular attraction between reducing sugar and copolymer PADB3.8 might be bigger than that between reducing sugar and copolymer PMDB , and then produced forces to drive reducing sugar into the top phase. Interactions including hydrophobic interactions, hydrogen bonds, charge effect determine the partitioning of product.
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Fig. 2. (a) Effects of salts and chaotropic agents on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentrations of all the salts were from 10 to 90 mM with 10 mM step. The partition coefficient was measured at 50 ◦ C. (b) Effects of temperature on partition coefficient of reducing sugar. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The partition coefficient of reducing sugar with 40 mM Na2 SO4 , NH4 Cl and (NH4 )2 SO4 in PADB3.8 /PMDB ATPS was measured at different temperature, respectively.
Microcrystalline cellulose as substrate in the study was insoluble, so it was completely partitioned in the bottom phase with immobilized cellulase in PADB3.8 /PMDB ATPS due to larger density of particles than that of water phase or large surface of the particles. In contrast to the soluble molecules, insoluble particulates partition between two phases and the interface, and with increasing size between on phase and the interface. The excess of substrate would be distributed in interface between two phases. Similar observation was previously reported [10]. The effect of temperature on partition coefficient of reducing sugar was shown in Fig. 2(b). The partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS showed a slight increase with the increasing of temperature. Partition coefficient of reducing sugar with 40 mM (NH4)2 SO4 was 2.09 at 30 ◦ C, and it increased to 2.61 when the temperature increased to 60 ◦ C. One possible explanation
was that the increasing temperature could increase the thermal motion of partitioning substance and the interactions between partitioning substance and copolymers. Therefore, the partition coefficient of reducing sugar had a slight increase when the temperature increased. 3.4. Biodegradation reaction of microcrystalline cellulose 3.4.1. Effects of reaction temperature on the cellulose biodegradation The hydrolysis of cellulose had been carried out in PNB /PMDB ATPS in previous work according to Liu’s publication [22]. Liang reported the optimum reaction temperature for cellulose biodegradation was 50 ◦ C [4]. However, the experiment in PNB /PMDB ATPS could only be carried out under 30 ◦ C because of the limitation
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when the temperature was above 50 ◦ C. Therefore, saccharification yield reached the maximum value at 50 ◦ C and 50 ◦ C was chosen as the optimum reaction temperature for cellulose biodegradation in the study. In summary, the novel PADB3.8 /PMDB ATPS showed a promising application in biodegradation of cellulose because it solved the problem of temperature limitation of thermo- response copolymer PNB in PNB /PMDB ATPS.
Fig. 3. Effects of reaction temperature on bioconversion of microcrystalline cellulose. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The biodegradation reaction was carried out in a shaker with 150 rpm at 30 ◦ C, 35 ◦ C, 40 ◦ C, 45 ◦ C, 50 ◦ C, 55 ◦ C, 60 ◦ C, respectively. The concentration of cellulose was 0.5% (w/v). The sample was taken out from top and bottom phases every 6 h to determine the concentration of reducing sugar until 72 h.
of thermo- response copolymer PNB . Therefore, yield of saccharification was limited and results were not quite satisfied. In this study, temperature limitation during biodegradation of cellulose in ATPS was solved. Novel pH–pH recyclable ATPS consisted of two pHresponse copolymers PADB3.8 and PMDB could be applied at higher temperature. Cellulase was immobilized on pH-response copolymer PMDB . The pH stability and thermo stability of free cellulase were improved by immobilizing cellulase on copolymer PMDB . The optimum pH and temperature of the pH-response copolymer PMDB with immobilized cellulase was also reported by Liang [4]. The effect of reaction temperature on the bioconversion in PADB3.8 /PMDB ATPS was firstly investigated. Fig. 3 shows the effect of reaction temperature on biodegradation of cellulose from 30 ◦ C to 60 ◦ C. From the results, it could be seen that saccharification yield increased sharply until 54 h at different reaction temperature, while after 54 h, saccharification yield maintained stable. With the reaction temperature increased from 30 ◦ C to 50 ◦ C, saccharification yield increased from 40.71% to 70.57% after 54 h. It could be concluded that the novel PADB3.8 /PMDB ATPS have obvious advantages over PNB /PMDB ATPS. Therefore, saccharification yield was improved greatly in PADB3.8 /PMDB ATPS after remove temperature limitation. When the reaction temperature went on increasing from 50 ◦ C to 60 ◦ C, saccharification yield decreased from 70.57% to 60.85% after 54 h. Inactivation of cellulase at such high temperature may lead to the decrease in saccharification yield. The thermal stability of PMDB with immobilized cellulase was reported in Liang’s publication [4]. It demonstrated that the absolute activity of immobilized cellulase decreased more than 30% when the temperature increased from 50 ◦ C to 60 ◦ C. Similar observations were also reported in Ince’s publication [23] and Dincer’s publication [24]. Ince et al. [23] investigated the effect of temperature on the catalytic activity of immobilized cellulase. It suggested that the optimum temperature for cross-linked cellulase was 50 ◦ C. The relative activity of cellulase increased obviously from 15 ◦ C to 50 ◦ C and decreased a lot from 50 ◦ C to 75 ◦ C. Dincer and Telefoncu [24] also demonstrated that the remaining activity of immobilized cellulase decreased a lot
3.4.2. Effects of initial substrate concentration on the cellulose biodegradation The effect of initial substrate concentration on cellulose biodegradation reaction was shown in Fig. 4(a). For the PADB3.8 /PMDB ATPS, the initial concentration of microcrystalline cellulose was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The reaction temperature was chosen 50 ◦ C. It could be seen that the concentration of substrate had obvious effect on saccharification yield. From the results, saccharification yield could reach 70.57% when the substrate concentration was 0.5% (w/v). The value was 5.72% higher than the reaction with 0.25% (w/v) substrate and 19.79% higher than the reaction with 1% (w/v) substrate. An increase of substrate concentration from 0.5% (w/v) to 1% (w/v) resulted in relatively obvious decrease of cellulose biodegradation. The marked decrease in saccharification yield at high substrate concentration (1% (w/v)) resulted in an optimum substrate concentration (0.5% (w/v)). It could be seen from Fig. 4(b) that the concentration of reducing sugar significantly increased from 3.92% (w/v) to 5.65% (w/v) linearly when the substrate concentration increased from 0.5% (w/v) to 1% (w/v). The concentration of the released reducing sugar could even reach 6.05% (w/v) at high substrate concentration (1% (w/v) substrate) after 72 h. Such high concentration of reducing sugar may also lead to the decrease in reaction rate because of product inhibition. Andrew et al. [25] used the model to explore various potential rate-limiting phenomena during cellulose biodegradation, such as substrate accessibility, product inhibition and the molecular weight of the cellulose substrate. Although cellulose was insoluble in this experiment, substrate concentration had obvious effect on cellulose biodegradation. Too low initial substrate concentration might not be enough for cellulase biocatalytic reaction, while too high initial substrate concentration in the reaction medium might limit the yield of saccharification due to decreasing mobility of insoluble substrate in the aqueous phase of the reaction mixture or increasing the viscosity of the reaction mixture which had an adverse effect on the mobility of the reactants and the release of products. Similar observations were also previously reported [26–28]. 3.4.3. Cellulose biodegradation in PADB3.8 /PMDB ATPS and in the single aqueous phase Cellulase was immobilized on copolymer PMDB before reaction. Biodegradation reactions in PADB3.8 /PMDB ATPS with immobilized cellulase were carried out in the experiment. ATPS was composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. Biodegradation with immobilized cellulase in the single aqueous phase was conducted as a control experiment. Single aqueous phase used for biodegradation reaction was composed by 2.5% (w/v) PMDB which immobilized cellulase on. Both reactions were carried out in a shaker at 150 rpm (50 ◦ C). Initial substrate concentration was 0.5% (w/v). Fig. 5 shows the comparison of two reactions carried out in PADB3.8 /PMDB ATPS and in the single aqueous phase. In PADB3.8 /PMDB ATPS, insoluble substrate and immobilized cellulase were biased to bottom phase, and product was partitioned to top phase in presence of 40 mM (NH4)2 SO4 . Microcrystalline cellulose was catalyzed into reducing sugar, and products entered into the top phase. In this way, inhibition of products was removed and
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Fig. 5. Bioconversion of cellulose in ATPS and in single aqueous phase. Cellulase was immobilized on copolymer PMDB . The initial concentration of substrate was 0.5% (w/v). The reaction of biodegradation of microcrystalline cellulose was carried out in PADB3.8 /PMDB ATPS and single aqueous phase in a shaker at 50 ◦ C. PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS.
Fig. 4. (a) Effects of initial substrate concentration on bioconversion of cellulose. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The biodegradation reaction was carried out in a shaker with 150 rpm at 50 ◦ C. The sample was taken out from top and bottom phases every 6 h to determine the concentration of reducing sugar until 72 h. (b) The concentration of reducing sugar with different initial substrate concentration. Cellulase was immobilized on copolymer PMDB . PADB3.8 /PMDB ATPS were composed by 2.5% (w/v) PADB3.8 and 2.5% (w/v) PMDB with immobilized cellulase. pH of ATPS was adjusted to 5.5 by dropwise adding 150 mM NaOH solution. The concentration of (NH4)2 SO4 was 40 mM in PADB3.8 /PMDB ATPS. The initial concentration of substrate was 0.25% (w/v), 0.5% (w/v), 0.75% (w/v) and 1% (w/v), respectively. The biodegradation reaction was carried out in a shaker with 150 rpm at 50 ◦ C.
saccharification yield was improved in ATPS compared with that in the single aqueous phase. The saccharification yield and reducing sugar concentration in ATPS were 74.34% and 4.13% (w/v) after 72 h, respectively. While the values in the single aqueous phase were 65.47% and 3.64% (w/v), respectively. The accumulated products in single aqueous phase during batch reaction inhibited hydrolytic rate. The similar observations were also emphasized by Andric et al. [9] and Gan et al. [29]. The saccharification yield in PADB3.8 /PMDB
ATPS was improved greatly compared with the result of previous work in PNB /PMDB ATPS [22]. Although the increase of saccharification yield was limited because of low partition coefficient of product, these novel recyclable ATPS certainly had advantages in industrial production because the new systems solved the problem of product inhibition during biocatalytic reaction and saccharification yield in PADB3.8 /PMDB ATPS was improved compared with the reaction in the single aqueous phase. The final saccharification yield still had a large space for improvement. We are now still looking for other salts to improve the partition coefficient of reducing sugar in PADB3.8 /PMDB ATPS. If the partition coefficient could be increased much more, saccharification yield should also be increased much higher. Furthermore, many other bioconversion reactions could also be carried out in this novel recyclable ATPS not limited to cellulose biodegradation. Product inhibition could also be solved and products yield would be increased compared with that in the single aqueous phase.
Fig. 6. Reusability of immobilized enzyme in recyclable PADB3.8 /PMDB ATPS. The reaction was carried out in a shaker with 0.5% (w/v) substrate at 50 ◦ C.
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3.4.4. Reusability of immobilized cellulase in PADB3.8 /PMDB ATPS After biodegradation reaction, both copolymers and immobilized cellulase could be recovered with high recovery and low cost by aforementioned method. The saccharification yield could still reach 66.15% after immobilized cellulase was recycled five times in recyclable PADB3.8 /PMDB ATPS (Fig. 6). Possible factors causing the decrease of saccharification yield included the loss of immobilized cellulase during polymer recovery process and unavoidable enzyme deactivation during centrifuge separation and biodegradation reaction. 4. Conclusions In this study, novel recyclable PADB3.8 /PMDB ATPS with watersoluble immobilized cellulase were used to biodegradation of cellulose. Cellulase was immobilized on PMDB , with over 80% reserved enzyme activity. The recycling recoveries of both copolymers were over 95%. A significant advantage is that phase-forming copolymers and cellulase could be recycled at a very low cost for many times. On the other hand, phase transfer bioconversion of cellulose has been carried out in PADB3.8 /PMDB ATPS. The saccharification yield in ATPS could reach 70.57% after 54 h, 9.3% higher than that in the single aqueous phase. The saccharification yield could reach 66.15% after immobilized cellulase was recycled five times in ATPS. The yield of saccharification in PADB3.8 /PMDB ATPS had been improved greatly compared with the result of previous work in PNB /PMDB ATPS. The novel PADB3.8 /PMDB ATPS could also be applied to other phase transfer bioconversion. References [1] D. Ciolacu, S. Gorgieva, D. Tampu, V. Kokol, Enzymatic hydrolysis of different allomorphic forms of microcrystalline cellulose, Cellulose 18 (2011) 1527–1541. [2] R. Smith, Biodegradable Polymers for Industrial Applications, Woodhead Publishing, London, 2005. [3] N. Ortega, M.D. Busto, M. Perez-Mateos, Kinetics of cellulose saccharification by trichoderma reeseri cellulases, International Biodeterioration and Biodegradation 47 (2001) 7–14. [4] W.J. Liang, X.J. Cao, Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization, Bioresource Technology 116 (2012) 140–146. [5] Y. Zhang, J.L. Xu, D. Li, Z.H. Yuan, Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100, Biocatalysis and Biotransformation 28 (2010) 313–319. [6] J.Q. Zhou, Immobilization of cellulase on a reversibly soluble-insoluble support: properties and application, Journal of Agricultural and Food Chemistry 58 (2010) 6741–6746. [7] M.A. Abd El-Ghaffar, K.S. Atia, M.S. Hashem, Synthesis, Characterization of binary copolymers of methyl methacrylate with glycidyl methacrylate and 2hydroxy ethyl methacrylate as carriers for cellulase, Journal of Applied Polymer Science 117 (2010) 629–638. [8] Y.J. Han, H.Z. Chen, Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase, Bioresource Technology 102 (2011) 4787–4792.
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[9] P. Andric, A.S. Meyer, P.A. Jensen, K. Dam-Johansen, Reactor design for minimizing product inhibition during enzymatic lignocelluloses hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes, Biotechnology Advances 28 (2010) 308–324. [10] P.A. Albertsson, Partition of Cell Particles and Macromolecules, Third ed., John Wiley & Sons, Inc., Canada, 1986, pp. 1–110. [11] O. Aguilar, M. Rito-Palomares, Aqueous two-phase systems strategies for the recovery and characterization of biological products from plants, Journal of the Science of Food and Agriculture 90 (2010) 1385–1392. [12] R. Dembczynski, W. Bialas, T. Jankowski, Recycling of phase components during lysozyme extraction from hen egg white in the EO50 PO50 /K2 HPO4 aqueous twophase system, Biochemical Engineering Journal 51 (2010) 24–31. [13] F.C. de Oliveira, J.S.R. Coimbra, L.H.M. da Silva, E.E.G. Rojas, M.C.H. da Silva, Ovomucoid partitioning in aqueous two-phase systems, Biochemical Engineering Journal 47 (2009) 55–60. [14] M. Canales, C. Ballesteros, J.A. Moreno-Cid, A.M. Espinosa, M. Villar, J. de la Fuente, Extractive bioconversion to produce the Aedes albopictus akirin in an aqueous two-phase system supporting Pichia pastoris growth and protein secretion, Biochemical Engineering Journal 46 (2009) 105–114. [15] X. Li, Z. Lian, B. Dong, Y. Xu, Q. Yong, S.Y. Yu, Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG6000/sodium citrate aqueous two-phase system, Chemical Engineering Journal 28 (2011) 1897–1901. [16] W. Wang, J.F. Wan, B. Ning, J.N. Xia, X.J. Cao, Preparation of a novel lightresponse copolymer and its application in recycling aqueous two-phase systems, Journal of Chromatography A 1205 (2008) 171–176. [17] B. Ning, J.F. Wan, X.J. Cao, Preparation and recycling of aqueous two-phase systems with pH-response amphiphilic terpolymer PADB , Biotechnology Progress 25 (2009) 820–824. [18] S. Miao, J.P. Chen, X.J. Cao, Preparation of a novel thermo-response copolymer forming recyclable aqueous two-phase system and its application in bioconversion of penicillin G, Separation and Purification Technology 75 (2010) 156–164. [19] H. Li, X.J. Cao, Bioconversion of cephalosporin-G to 7-ADCA in a pH-thermo sensitive recycling aqueous two-phase systems, Process Biochemistry 46 (2011) 1753–1758. [20] T.K. Ghose, Measurement of cellulase activities, Pure and Applied Chemistry 59 (1987) 257–268. [21] J. Szczodrak, The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol, Biotechnology and Bioengineering 32 (1988) 771–776. [22] J.J. Liu, X.J. Cao, Biodegradation of cellulose in novel recyclable aqueous twophase systems with water-soluble immobilized cellulase, Process Biochemistry 47 (2012) 1998–2004. [23] A. Ince, G. Bayramoglu, B. Karagoz, B. Altintas, N. Bicak, M.Y. Arica, A method for fabrication of polyaniline coated polymer microspheres and its application for cellulase immobilization, Chemical Engineering Journal 189-190 (2012) 404–412. [24] A. Dincer, A. Telefoncu, Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads, Journal of Molecular Catalysis B: Enzymatic 45 (2007) 10–14. [25] J.G. Andrew, J.S. Jonathan, J.L. James, A Mechanistic Model for Enzymatic saccharification of cellulose using continuous distribution kinetics II: cooperative enzyme action, solution kinetics, and product inhibition, Biotechnology and Bioengineering 109 (2012) 676–685. [26] A.F. Abdel-Fattah, M.A. Abdel-Naby, Pretreatment and enzymic saccharification of water hyacinth cellulose, Carbohydrate Polymers 87 (2012) 2109–2113. [27] M. Chen, J. Zhao, L.M. Xia, Enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars, Carbohydrate Polymers 71 (2008) 411–415. [28] Y. Zheng, Z.Y. Pan, R.H. Zhang, D.H. Wang, Enzymatic saccharification of dilute acid pretreated saline crops for fermentable sugar production, Applied Energy 86 (2009) 2459–2465. [29] Q. Gan, S.J. Allen, G. Taylor, Kinetic dynamics in heterogeneous enzymatic hydrolysis of cellulose: an overview, an experimental study and mathematical modeling, Process Biochemistry 38 (2003) 1003–1018.