Batch Hydrolysis and Rotating Disk Membrane Bioreactor for the

Article pubs.acs.org/IECR

Batch Hydrolysis and Rotating Disk Membrane Bioreactor for the Production of Galacto-oligosaccharides: A Comparative Study Dwaipayan Sen, Ankur Sarkar, Saikat Das, Ranjana Chowdhury, and Chiranjib Bhattacharjee* Department of Chemical Engineering, Jadavpur University, Kolkata-700032, India ABSTRACT: Galactosyl-oligosaccharide (GOS) is a prebiotic carbohydrate, produced from lactose hydrolysis, that serves as a value-added functional food element for humans. Now GOS can be produced through enzymatic reaction with lactose either in batch mode or with immobilized enzyme on membrane. In the second case, the main concern is the fouling of the membrane that could reduce both the GOS yield and purity. The present study is thus an attempt to project the superiority of a membrane reactor called the rotating disk membrane bioreactor (RDMBR) over batch mode to obtain purified GOS with high yield. It was found that GOS yield and purity were 32.4% and 77% respectively in batch mode followed by diafiltration-assisted nanofiltration. However, in the immobilized state they were 67.4% and 80.2% at 105 rad s−1 membrane speed. Retention of monosaccharides that inhibit enzyme in the reaction volume of batch mode reduced the yield of GOS. On the contrary, simultaneous production and purification of GOS in RDMBR led to a high yield of GOS.

1. INTRODUCTION Nowadays consumers are more concerned about health issues and consume products that can provide good nutrition to their health, called functional foods. One of these functional foods is prebiotic carbohydrates that can beneficially affect the host by selectively stimulating the growth of bacteria in the colon. Galacto-oligosaccharides (GOS) are one such prebiotic element that can be produced from lactose present in abundance in milk. Extensive research was carried out for better utilization of lactose, constituting 70% of the dissolved solids in milk.1 Yang and Silva2 suggested several promising areas for continuing research on the properties of lactose and various whey products based on lactose. Lactose is generally considered as an undesirable product present in the milk, because of its intolerance by many people, low solubility, and tendency to crystallize in water at low temperatures.3,4 This limitation of lactose encouraged many researchers to develop a technology for converting it into oligosaccharides, such as GOS, which is a highly beneficial and desirable prebiotic ingredient to have present in dairy-based foods.5 GOS, mainly produced from lactose hydrolysis either by acidic treatment or by enzymatic method, stimulate the proliferation of bifidobacteria and lactobascilli in the intestine, leading to an improvement of intestinal microflora.1,6,7 Acid hydrolysis of lactose leads to a decrease in the nutritional values of the milk products with an undesirable generation of color and odor, making the process less attractive to the makers.8,9 However enzymatic hydrolysis, aided by β-galactosidase (EC 3.2.1.23) enzyme, alleviates the problems associated with the acid hydrolysis. In an enzymatic process, β-galactosidase catalyzes the hydrolysis reaction as a forward reaction and oligosaccharides synthesis through a transgalactosylation reaction.10 In the last few decades, much research has been carried out on GOS production from lactose. Iwasaki et al.1 investigated the production of GOS in batch hydrolysis of lactose at 313 K and pH 4.5 using β-galactosidase extracted from Aspergillus oryzae along with the formulation of a predictive model for GOS production. They had shown in their study that increasing © 2012 American Chemical Society

lactose concentration favors the transgalactosylation reaction and hence the production of GOS. Commercially the hydrolysis reaction is carried out in batch mode to maintain proper sterile conditions with an overall control of the regulatory parameters to obtain optimum GOS yield. However with this technique, the purity of GOS is mainly affected by the presence of enzymes, monosaccharides, and unreacted lactose present in the solution. Compared to this, the enzyme immobilization technique is another method for enzymatic hydrolysis of lactose to produce GOS. Especially hydrolysis using immobilized enzyme on some support offers a number of advantages over batch hydrolysis in terms of reusability of the enzyme, retention of the catalytic activity, and the cost reduction of the downstream process in addition to the production of good product quality.11 Selection of nanofiltration (NF) membrane as a support for enzyme immobilization offers an additional advantage of simultaneous production and separation of GOS from other low molecular weight sugars. Hence, amendment of the membrane separation technique, along with the conventional approach of lactose hydrolysis, ameliorates the production process of purified GOS.12 Microfiltration (MF) and ultrafiltration (UF) are two established membrane separation processes in the field of biotechnology as a means for the purification of oligosaccharides from high molecular weight enzymes and other saccharides.5,13−15 However, with the reaction mixture comprising GOS along with other low molecular weight sugars, MF or UF cannot effectively separate GOS from the reaction mixture. Nanofiltration (NF), aided by suitable molecular weight cutoff (MWCO) membrane, was proved to be a better alternative to MF and UF for the separation of GOS from low molecular weight mono- and disaccharides,16 enriching the purity of GOS. Received: Revised: Accepted: Published: 10671

March 6, 2012 June 10, 2012 July 21, 2012 July 21, 2012 dx.doi.org/10.1021/ie3005786 | Ind. Eng. Chem. Res. 2012, 51, 10671−10681

Industrial & Engineering Chemistry Research

Article

Especially application of NF is an attractive separation process for both desalting and concentration of UF permeate collected in dairy industry. This ensures the recovery of lactose in abundance for producing GOS.17 Basically, NF is a pressuredriven membrane separation process for the removal of ions, as in the case of production of whey and milk UF permeate. Suárez et al.18 investigated an additional effect of discontinuous diafiltration on NF to demineralize whey and milk to understand the superiority of diafiltration over single batch NF during the recovery of the desired components. They found that there was almost a 37−38% increase in the degree of ion removal with discontinuous diafiltration compared to the batch one without diafiltration. Bowen and Mohammad19 made a similar study on NF amalgamated with diafiltration in order to develop a predictive model based on extended Nernst−Plank equation to investigate the performance of a NF membrane in separating the components of a dye/salt solution. Especially adaptation of diafiltration is now state-of-the-art in the food, biotech, and pharmaceutical industries to enhance the purity of the desired components on the retentate side, without having so much mechanical effort to enhance the permeation of the low molecular weight components through membrane.20 In spite of all the promising perspectives of NF, there are some unresolved problems associated with NF, which limits the application of NF in large-scale industries.21 In any membrane separation process, one of the major associated drawbacks is membrane fouling. This is more pronounced in case of NF because of the intense interactions between the solutes and the membrane. The fouling increases at the nanoscale, which is difficult to understand.22,23 However the complexity of fouling is more pronounced with a membrane bioreactor (MBR), which is now a frequently used tool in large-scale industry for wastewater treatment.24 Moritz et al.25 studied a dead-end filtration module with charge distribution on the membrane surface to enhance the filtration performance of the NF membrane. Thanh et al.26 proposed the concept of a baffled membrane separation unit to make a considerable reduction in the cost of a membrane separation process due to the membrane fouling that resulted because of organic loading on the membrane unit. Still many of the studies have not addressed the fouling issue for the membrane separation process, especially under immobilized conditions. In this study an attempt was made to develop the concept of a rotating disk membrane bioreactor (RDMBR) to alleviate the problem of membrane fouling associated with a membrane bioreactor, after giving rotational motion to the membrane. With this setup an investigation was made here to understand the purity achieved for GOS during its production by enzymatic hydrolysis of lactose aided by immobilized β-galactosidase enzyme on NF membrane. Additionally, the effect of diafiltration on NF for the effective separation of GOS produced by batch hydrolysis was also studied with a high-shear device called a rotating disk membrane module (RDMM). Finally, a comparative outlook was given in the present study to understand the superlative nature of the two approaches based on NF for the production of GOS with utmost purity and yield.

Table 1. Membrane Specifications membrane

MWCO, kg mol−1

TFC-SR2

0.4

Vivaflow 200

50

specifications • 9.8% NaCl rejection • pH 4−9 • maximum operating pressure 3.45 MPa • maximum process temperature 318 K • active surface area 0.0025 m2 • overall length/height/width 0126/0.138/0.038 m • channel width/height 0.01/0.0004 m • pH 2−14 • maximum operating pressure 0.4 MPa • active surface area 0.02 m2

2.2. UF Membrane. Polyether sulfone (PES) UF membrane (50 kg mol−1), purchased from Vivascience AG, Germany, was casted in a housing module called Vivaflow 200, supplied by the same manufacturer. Detailed specifications for this module are presented in Table 1. 2.3. Chemicals. β-Galactosidase enzyme (market name Biolacta FN5; EC 3.2.1.23) extracted from Bacillus circulans, with initial lactose activity, 4500 LU g−1, was procured from Burra Foods, Australia. [1 LU (abbreviated form of “lactose unit”) is defined as the amount of enzyme required to produce 1.67 × 10−11 kmol of glucose/s from lactose.28] Lactose was purchased from SISCO Research Laboratory Pvt. Ltd., Mumbai, India. Polyethyleneimine [500 kg m−3 (50% w/v), average molecular weight 750 kg mol−1] and glutaraldehyde [250 kg m−3 (25% w/v) of aqueous solution] were purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. The commercial enzyme preparation has a protein content of 0.041 kg per kg of solid powder. Enzymatic protein content was measured by the Folin−Lowry29 method using Folin− Ciocaltaeu’s phenol reagent (2 N, AR grade) supplied by SISCO Research Laboratory Private Limited, Mumbai, India. Hydrochloric acid, sodium carbonate, caustic soda, anhydrous copper sulfate, and sodium potassium tartarate were procured from Merck India Ltd., Mumbai, India. 2.4. Analytical Instruments. Deionized (DI) water was collected from an ultrapure water system (model Arium 611) from Sartorius AG. The feed for deionized water system was distilled water from a reverse osmosis (RO) system (model Arium 61315, Sartorius AG). A Varian UV−visible spectrophotometer (Cary50 Bio) was used for protein concentration measurement at 750 × 10−9 m (750 nm). HPLC (PerkinElmer, Series 200) with RI detector and Spheri 5 amino column (5 × 10−6 m, 0.046 × 0.220 m)30 was used with a mobile phase of 75:25 (v/v) acetonitrile:water at a flow rate of 8.33 × 10−9 m s −1 (0.5 mL min−1) for sugar analysis. The concentration of lactose, monosaccharides, and GOS was measured from the standard curves31 prepared after calibrating with known standard solutions of lactose, glucose, galactose, and raffinose. However, one of the typical advantages obtained using a RI detector in HPLC analysis of carbohydrates is the detector shows identical response for all carbohydrates at the same concentration. Therefore, the calibration curves for all individual pure carbohydrates, showing the detector response variations with the concentrations, will possess the same correlations for standard curves between the detector response and the concentration. During all the chromatographic runs, column oven temperature was maintained constant at 298 K. Field emission scanning electron microscope (FESEM) (Quanta Cryo Environmental Scanning EM) images were graciously done by Bio21 Institute, Melbourne, Victoria, Australia.

2. MATERIALS SPECIFICATIONS 2.1. NF Membrane. TFC-SR2 membrane was procured from Koch Membrane Systems (San Diego, CA). According to the manufacturers, these membranes are a polyamide thin-film composite with a microporous polysulfone supporting layer.27 Table 1 presents the properties of TFC-SR2 membrane. 10672

dx.doi.org/10.1021/ie3005786 | Ind. Eng. Chem. Res. 2012, 51, 10671−10681


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Figure 1. Schematic diagram of the experimental setup for the production of purified GOS obtained from batch hydrolysis of lactose.

2.5. Membrane Compaction and Water Run.32 Prior to the experimental run both NF membrane and UF cross-flow membrane were subjected to compaction for about 1 h with DI water at a pressure of 1.4 and 0.9 MPa, respectively, higher than that of the highest operating pressure to prevent any possible changes in the membrane pore diameter during filtration. Once the water flux became steady with no further decrease with time, it was concluded that full compaction of the membrane had taken place. Both UF and unimmobilized NF membranes were washed thoroughly with distilled water followed by chemical wash after every run to remove any deposited fouling layer. The water fluxes obtained from such studies were found to be within 2% of the initial water flux, thus showing minimum fouling resulting from the proposed separation scheme.

ensured with this arrangement of experimental planning. One is the proper contact between the substrate and enzyme, and the other one is the ease in the permeation of low molecular weight sugars through the membrane to achieve high-purity GOS. Figure 2 shows a schematic diagram to present the arrangement made with the RDMBR. Details of both the arrangements will be discussed in the subsequent sections. 3.1. Batch Hydrolysis of Lactose and Purification of GOS. Lactose solution (100 kg m−3, 10% w/v) prepared in 0.1 M acetate buffer in order to maintain a pH value at 6.5 was incubated at a fixed temperature of 277 K for the entire reaction period after dosing with 0.1 kg m−3 (0.1 mg mL−1) of β-galactosidase enzyme extracted from B. circulans. This reaction mixture was kept with a mild stirring for 5 h (18 000 s) to ensure an effective contact between the substrate and the enzyme. Lactose solution (100 kg m−3, 10% w/v) was considered on the basis of the free enzymatic reaction to produce GOS from lactose using β-galactosidase from B. circulans.31 However, in a particular study by Das et al.,31 the maximum concentration of GOS was achieved with 200 kg m−3 (20% w/v) of lactose solution. In the current work, GOS synthesis in RDMBR was observed with a lactose concentration of 200, 100, and 50 kg m−3, resembling the achievable concentration of GOS in the study made by Das et al.31 However, with 200 kg m−3 (20% w/v) concentration of lactose in RDMBR, the GOS production ceased after a certain time interval; this is attributable to concentration polarization because the large amount of initially produced GOS resists the convective diffusion of lactose toward the enzyme, immobilized on the membrane. At 100 kg m−3 (10% w/v) the maximum GOS production was found in the membrane reactor, perhaps because of less resistance to the substrate diffusion toward the enzyme bed. Each of the experimental runs was replicated three times to ensure the reproducibility of the experiments with a

3. EXPERIMENTAL SECTION In compliance with the objective of the present study, experiments were segregated into two segments. In one part, batch hydrolysis of lactose was performed to produce GOS and fed to the 50 kg mol−1 UF membrane followed by the 0.4 kg mol−1 NF membrane fitted with RDMM in tandem. Figure 1 presents an illustrative schematic representation of the batch hydrolysis encompassing the other subsequent steps. To achieve maximum purity of GOS, diafiltration was done during the NF membrane separation step. In another part, the concept of RDMBR was introduced, where simultaneous production along with the separation of GOS can be achieved. In this setup, immobilization of enzyme was carried out on the NF membrane. A mild rotation was given to the membrane in order to emulate the phenomenon of rotating contactors and thus to create some turbulence in order to break the concentration boundary layer developed in the vicinity of the immobilized membrane. Thus, two primal objectives were 10673



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Figure 2. Schematic diagram of rotating disk membrane bioreactor.

of β-galactosidase enzyme on NF membrane following the method cited by Isgrove et al.34 Immobilization on membrane involved some major steps. First, acid hydrolysis of the amide groups on the membrane surface was done to generate amine groups, followed by the formation of a covalent bond between the amine and the aldehyde group of gluteraldehyde. Finally, an attempt was made to facilitate the formation of cross-linked enzyme aggregates (CLEA) using gluteraldehyde (GA), PEI, and enzyme with the membrane surface. The detailed protocol for this immobilization of enzyme is given below. Primarily, the membrane surface was incubated for 2 h (7200 s) at room temperature with 2.9 M hydrochloric acid (HCl) and washed thoroughly with 0.1 M phosphate buffer (pH 8). Now the membrane was placed in 10 mL (0.000 01 m3) of 25 kg m−3 (2.5% w/v) gluteraldehyde solution prepared in 0.1 M phosphate buffer (pH 8) for 15 min (900 s) at 293 K. Again the membrane was rinsed with deionized water and incubated in 1% (v/v) PEI prepared in 0.1 M phosphate buffer (pH 8) for 1 h (3600 s) at room temperature followed by a thorough wash with water. Again the membrane after PEI attachment was placed in gluteraldehyde solution as done before. Finally, 10 mL (0.000 01 m3) of 5 kg m−3 (5 mg mL−1) of enzyme solution was given on the treated membrane and incubated for 2 h (7200 s) followed by a water wash. The immobilized membrane was stored in 0.1 M phosphate buffer (pH 8) until use. Figures 3−5 show FESEM images of TFC-SR2 membrane under different conditions. Parts a and b of Figure 3 respectively show the cross-sectional and top view of the NF membrane used in this work. FESEM images of NF membrane (magnification of 4000× and 24 000×, respectively) show that these membranes are densely intermeshed, significantly different from the fiberlike ultrafiltration membrane, and thereby manifests a strong support for adsorption-based fouling or

deviation of 4−5% of the mean value. Therefore, complying with the results found with 100 kg m−3 (10% w/v) lactose solution fed to the membrane reactor, the batch reaction followed by NF in diafiltration mode for the purification of GOS was performed. Finally, a comparative performance evaluation with these two modes of reaction was carried out in order to conclude the percentage yield and purity of the GOS. After 5 h (18 000 s) the carbohydrates’ solution was withdrawn from the reactor and was fed to a 50 kg mol−1 UF cross-flow membrane to separate enzyme from the product. A transmembrane pressure (TMP) of 0.15 MPa and volume concentration factor (VCF) of 4 (eq 1) were maintained in the membrane separation process. VCF =

initial feed volume final retentate volume

(1)

After separation of the enzyme using membrane, the activity of the retained enzyme on the retentate side was found by activity assay28 to decide the reusability of the enzyme solution in the subsequent batch processes. Moreover, no permeation of the enzyme through 50 kg mol−1 UF membrane was ascertained by a Folin−Lowry29 protein assay of the collected permeate. This permeate, comprising of mainly GOS, monosaccharides, and lactose, was again fed to the RDMM equipped with NF membrane in conjunction with the threestage discontinuous diafiltration, varying the membrane speeds at 0, 42, 105 rad s−1, in order to separate GOS from mono- and disaccharides. Separation was carried out at 1 MPa transmembrane pressure (TMP) maintaining a constant temperature of 298 K. 3.2. Lactose Hydrolysis Using Immobilised Enzyme on NF Membrane. Polyethyleneimine (PEI), an extremely branched cationic chain polymer,33 was used for immobilization 10674



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Figure 3. FESEM images of TFC-SR2 membrane NF membrane: (a) cross-sectional image and (b) front image.

Figure 5. FESEM image of enzyme immobilized TFC-SR2 membrane NF membrane after completion of experimental run for a particular membrane speed. (Image was taken of static membrane.)

Figure 4. FESEM image of enzyme immobilized TFC-SR2 membrane NF membrane.

concentration polarization because of solute deposition. Figure 5 shows a vivid realization of carbohydrates’ fouling after the run. Figure 4 is significant in this particular study, showing the 12 000× magnified image of enzyme attachment with PEI chains on the membrane. The figure shows that some of the enzyme molecules seem to be not linked with the PEI chains and, possibly, are loosely bound to the membrane surface. These are possibly the adsorbed enzymes that were released even at low membrane speed. Detailed discussions on this can be found in the Result and Discussion section. After immobilization 100 kg m−3 of lactose solution in 0.1 M acetate buffer (pH 6.5) was fed to the immobilized membrane reactor and the reaction was carried out for 3 h (10800 s) at 277 K under an initial TMP of 1 MPa. The TMP was increased as the experiment proceeds to maintain the initial permeation flow rate of 3.4 × 10−8 m3 s−1 (around 2 mL min−1). The membrane speed was varying at 0, 21, 42, 63, 84, 105, 157, 209, and 314 rad s−1. For each of the membrane speeds the reaction period was 3 h (10800 s), in order to study

the rotation effect on the immobilization as well as purification of GOS. 3.3. Enzyme Activity Assay. Enzyme activity was determined according to the method suggested by Splechtna et al.28 with slight modifications. Briefly, 20 μL (2 × 10−8 m3) of enzyme solution was added to 480 μL (48 × 10−8 m3) of substrate solution, containing 600 mM (205.2 kg m−3) lactose in 50 mM (50 × 10−3 M) sodium phosphate buffer (pH 6.5). Incubation was continued for 10 min (600 s) at 303 K. Reaction was stopped by boiling the sample for 3 min (180 s). The amount of glucose released was measured by direct comparison of the reading with the standard curve in the plot of glucose concentration versus enzyme activity times the reaction time [kg m−3 versus kg m−3 s] obtained from free enzyme reactions.

4. RESULTS AND DISCUSSION To refine the excellence of a RDMBR the following discussions are augmented into two portions. The first part provides an 10675



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Figures 7−9 show the variation in the NF permeate flux with time at each stage of diafiltration for varying membrane rotational speed in RDMBR with a volume concentration factor (VCF) of 1.5. The figures show that there is an initial gain in the permeate flux until the second stage, attributable to an additional washing of the membrane surface because of dilution of the retentate portion during diafiltration. Moreover, with the increase in the membrane speed there was a substantial increase in the flux, conferring the alleviation of the concentration polarization layer in the vicinity of the membrane surface. However, at the third diafiltration stage, the initial permeate flux became low compared to the previous stage, even after the dilution of the retentate. Unfortunately, studies on the interactions between carbohydrate and NF membrane have not been done so far in a very extensive way. Therefore, it is hard to say the exact reason for this ambiguous behavior at the third stage of diafiltration is attributable to maximum dilution. It might be that the adsorption of microsolutes (monosaccharides and lactose) within the dense NF membrane interstices creates an obstruction to the flow, and hence, there is a substantial drop in the initial permeate flux. Moreover, in the case with the NF, this effect was much more pronounced compared to UF or MF, as nanoscale structure of the NF membrane makes the process complicated and unpredictable. Another reason might be because of the negative polarity resistances imparted against the permeation due to increased hydroxyl groups with a GOS concentration increase in the retentate.35 In the case with a membrane speed of 105 rad s−1, the first stage initial permeate flux was low enough compared to the second and third stages, which is quite significant. It is difficult to make any conclusive comments on this particular discrepancy observed with 105 rad s−1 membrane speed. Now polymeric NF membrane is generally a dense intermeshing of the polymeric fibers and is deformable under the applied stress. Because of this, perhaps high rotational speed induced enhanced shear stress between the concentrated feed medium and the membrane surface. Presumably this leads to a longitudinal deformation of the upper polymeric layer of the membrane surface and reduced the gaps between the interstices. This adverse effect of membrane rotation along with the concentration polarization ultimately reduced the initial permeate flux for highly concentrated feed media. However, with the subsequent dilution, shear stress and

overview of the production of GOS through batch hydrolysis of lactose followed by membrane separation. The second part is an elaboration on the RDMBR to produce GOS. Subsequently, after having a thorough discussion on the pros and cons of both of the processes an attempt has been made to elucidate the merits of RDMBR over the conventional batch process. 4.1. Batch Study of Lactose Hydrolysis. Figure 6 presents a comparative layout on the percentage yield of

Figure 6. Percentage yield of GOS and monosaccharides with time for 100 kg m−3 of lactose concentration at pH 6.5 and 277 K.

monosaccharides and GOS with time in the case with the batch hydrolysis of lactose at a temperature of 277 K and pH 6.5, where the percentage yield is defined as the carbohydrate percentage over the initial amount of lactose. The figure shows that the GOS yield curve after 7200 s almost forms an asymptote, making possible a conclusion on restricted GOS production. The observation thus confers a possible presumption of monosaccharide inhibition of that part of the enzyme which catalyzes the transgalactosylation reaction. However, an almost 7% increase in the monosaccharides’ yield compared to a 0.3% increase in GOS yield at 18 000 s thus preludes the possible shoot-up of the hydrolysis reaction.

Figure 7. Variation of permeate flux with time for three-staged diafiltration for purifying GOS using NF in RDMM with a membrane rotational speed of 0 rad s−1 and a volume concentration factor of 1.5 under the transmembrane pressure of 1 MPa. 10676



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Figure 8. Variation of permeate flux with time for three-staged diafiltration for purifying GOS using NF in RDMM with a membrane rotational speed of 42 rad s−1 and a volume concentration factor of 1.5 under the transmembrane pressure of 1 MPa.

Figure 9. Variation of permeate flux with time for three-staged diafiltration for purifying GOS using NF in RDMM with a membrane rotational speed of 105 rad s−1 and a volume concentration factor of 1.5 under the transmembrane pressure of 1 MPa.

concentration polarization effects became low, resulting in an effective increase in the initial permeate flux. Figure 10 shows the variation of GOS percentage purity, defined by eq 2, at different stages and membrane speeds. More likely, at higher membrane speed, increased dilution with subsequent diafiltration stages increases the purity of GOS. The figure shows that after the first stage of diafiltration there is a sharp increase in the purity, ensuring the maximum permeation of the lower molecular weight sugars through the membrane. Around 77% pure GOS was recovered at the second stage of the diafiltration for 105 rad s−1 membrane speed. percentage purity of GOS =

100 × mass of GOS present total mass of sugars present (2)

However, the yield of GOS after batch hydrolysis was 32.4%, attributable to a possible inhibition of enzyme due to the presence of monosaccharides in the reaction medium. Therefore, the presence of monosaccharides in the reaction medium ultimately had an adverse effect on the yield of GOS and requires the withdrawal of these monosaccharides from the

Figure 10. Variation of percentage purity of GOS with diafiltration stages for different membrane speeds. 10677



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with time the fouling layer was more pronounced with 84 rad s−1 membrane speed compared to 105 rad s−1, and therefore, at the end flux became higher at 105 rad s−1 compared to 84 rad s−1. However, at a higher membrane speed of 157 rad s−1 an almost 48% increase in the initial permeate flux was observed compared to 105 rad s−1. Considering the fact that membrane deformed at high speed, for which the flux became reduced, it can be said that above 157 rad s−1 apparently it looks like the covalently linked enzyme got disengaged from the surface, which lead to an enhancement of the permeate flux. However, the released enzyme in this case might be the adsorbed portions that were leached out from the surface due to the shear that developed at high membrane speed. Subsequent analysis of Figures 12 and 13 will guide us to a better depiction of the

reaction mixture. RDMBR, discussed in the next subsequent portion, is thus a unique membrane bioreactor module where one can use it both for the simultaneous production and purification of the product. Moreover, because of the mechanical movement of the membrane, it behaves as a high shear device that can alleviate the concentration polarization and ensure the proper enzyme−substrate contact for high productivity. 4.2. Immobilization Study of Lactose Hydrolysis. TCFSR2 is basically a thin composite film membrane, composed of polyamide−polysulfone and possessing a hydrophobic property. However, PEI cross-linked on the membrane surface during immobilization led to a surface modification, converting from a hydrophobic to a hydrophilic membrane surface with a 58% increase in the water flux.36 This could be considered as one of the advantages with PEI-aided immobilization on a hydrophobic surface. An immobilization yield of 82.6% was found after immobilizing β-galactosidase on the membrane surface. Figure 11 shows the variation of permeate flux with

Figure 12. Variation in the percentage enzyme detachment from the immobilized surface for different membrane rotational speeds in RDMBR.

Figure 11. Variation of permeate flux with time for different membrane speeds in RDMBR for 100 kg m−3 of lactose concentration at pH 6.5 and 277 K.

time at different membrane speeds. However, one of the key issues with this membrane module was the residence time of the lactose within the reaction volume, i.e., above the enzyme immobilized membrane surface. At an initial stage of the process in RDMBR the permeation of the solutes was much higher compared to the subsequent times. Therefore, the residence time of lactose was less in the beginning while at the end lactose/monosaccharides’ accumulation reduced the yield of GOS. To maintain a constant moderate permeation rate in the whole process for a particular membrane speed, TMP was adjusted. On average a very slow permeation flow rate was adjusted to 3.4 × 10−8 m3 s−1 (around 2 mL min−1) during the run time to increase the contact period between lactose and immobilized enzyme. With the increase in membrane speed, permeate flux increases because of the alleviation of the resistances on the membrane surface. Figure 11 shows that at a membrane speed of 84 rad s−1 the initial permeate flux is more than the initial flux obtained for 105 rad s−1, which can be explained by a similar concept based on longitudinal deformation of membrane provided in the previous section. But

Figure 13. Percentage yield and percentage purity of GOS with varying membrane speeds in RDMBR.

actual fact in this regard and thus help to confine our search for the optimum operating parameters. Figure 12 shows that more than 50% of the immobilized enzymes were detached from the membrane surface at 10678



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314 rad s−1 membrane speed, due to development of high rotational stress on the immobilized surface. Until the 157 rad s−1 membrane speed, the release of enzyme was well below 15%, which is quite satisfactory in the sense that the enzyme released here might be the adsorbed enzyme within the membrane surface instead of the covalently linked one. Above 157 rad s−1, the percentage release of enzyme shoots up to more than 20%, which might be due to the release of both adsorbed and immobilized enzyme. It was observed that with increasing membrane speed from 105 to 157 rad s−1, time weighted average flux (TWAF)37 (eq 3) was increased by almost 16%, whereas the increase from 84 to 105 rad s−1 shows a mere 0.1% increase in the TWAF.

substrate and immobilized enzyme, which ultimately increases the yield and purity of GOS until 84 rad s−1. However, after this speed, yield was reduced by almost 5% at 105 rad s−1. Subsequently, at 157 rad s−1 the yield was almost reduced by 11% from the maximum yield. Whenever enzymes are released from the immobilized state the yield can be decreased and at the same time more permeation will also reduce the lactose residence time on the membrane surface. Hence yield of GOS was decreased because of these results due to increased membrane speed. It seems that a mere 5% reduction at 105 rad s−1 was primarily due to the reduction in lactose residence time attributing to the release of the adsorbed enzyme that act as fouling. However, 11% reduction in the yield is possibly attributable to the loss of immobilization and, therefore, less lactose contact with the enzyme to initiate the reaction. Moreover, 5% reduction in the yield compared to 11% is admissible, especially when the purity of the GOS was concerned along with the enzyme inhibition because of monosaccharide retention on the reaction side. Thus, apparently 105 rad s−1 membrane speed can be considered an optimum point where a reasonable purity and a reasonable percentage yield for GOS were achieved.

t

TWAF =

∫t = 0 max J(t ) dt t

∫t = 0 max dt

(3)

tmax is the maximum run time, and J(t) is the permeate flux at any time t. Adsorbed enzyme either within the membrane pores or on the surface manifests a type of membrane fouling and will impart resistance to the permeation. TWAF comparison projects a clear vision on the membrane speed at which the resistance to permeation becomes lower. From the discussion the operability range for membrane speed should be in between 105 and 157 rad s−1 to ensure minimum fouling due to adsorption. However, data does not reflect whether the reduction in resistance was due to removal of adsorbed enzyme or undesirable release of immobilized enzyme. Figure 13, which shows the comparison between GOS purity and yield, apparently provides an idea of what happened with the increasing membrane speed. Figure 13 shows that the variation of percentage yield of GOS reaches a maximum (around 71%) at 84 rad s−1, whereas the purity of GOS is a maximum (around 85%) at 314 rad s−1. However, at 84 rad s−1 the purity was almost 8% less compared to 314 rad s−1. On the contrary, at 314 rad s−1 the yield of GOS was almost 36% less compared to 84 rad s−1. Increasing membrane speed results in better dispersion of the solute molecules throughout the feed solution and thus reduces the chances of concentration polarization layer formation near the membrane surface. This results in better contact between the

5. CONCLUSION The present paper is thus an attempt to investigate the novelty of RDMBR over batch hydrolysis of lactose followed by UF and NF of the reaction mixture in order to obtain pure GOS. Table 2 presents a comparative chart on the percentage yield and purity of GOS in both the membrane and batch reactor. Lactose hydrolysis using immobilized enzyme on the NF membrane surface in RDMBR was proved to be a better alternative, with more than 60% GOS yield, almost doubled compared to the GOS yield obtained in batch mode (32.4%). One of the basic problems associated with the batch mode is the reutilization of the enzyme. The separation of enzyme from the carbohydrate mixture obtained from the batch reaction using membrane could lead to the loss of enzyme and loss of its activity. On the contrary, RDMBR is a single setup where these problems associated with a batch mode reaction could be avoided, ultimately leading to a high yield with high purity of GOS at an optimum membrane speed of 105 rad s−1. Moreover, the proposed membrane reactor module here can serve a single operation for the production and purification of GOS, which ultimately reduces the downstream costs associated with individual unit operations. A GOS purity of 80% with a yield of around 67% was achieved with enzyme immobilized membrane (around 83% immobilization yield) fitted in a rotating membrane module with 100 kg m−3 lactose solution as feed to the reactor prepared in 0.1 M acetate buffer at pH 6.5. The temperature, maintained for this 3 h (10 800 s) reaction time, was 277 K with a constant permeate flux

Table 2. Comparative Figures of GOS Yield and Purity in Both Batch Mode and Immobilised Modea) mode

yield of GOS (%)

purity of GOS (%)

batch hydrolysis immobilised enzyme

32.4(1.0) 67.4(2.3)

77.0(2.1) 80.2(1.4)

a

Values in the parentheses indicate the standard deviation obtained for three observations.

Table 3. GOS Yield and Purity in Enzymatic Membrane Reactor Proposed Earlier proposed by Das et al.31 38

Engel et al. Pocedičová et al.39 Sen et al.40

a

membrane type

temp (K)

pH

yield of GOS (%)

Biolacta FN5

277

6.6

30.6

77

Maxilact L 2000 Maxilact LX 5000 Biolacta FN5

313 310 313

7.0 6.75 6.5

8.6 4.5 2.5 2.5 0.3

24 NAa NAa NAa NAa

enzyme name

50 kg mol−1 PES UF membrane followed by 0.4 kg mol−1 TFC-PA NF membrane 0.8 μ supported cross-linked PES membrane 150 kg mol−1 ceramic UF membrane 50 kg mol−1 PES UF membrane 5 kg mol−1 PES UF membrane 5 kg mol−1 CTA UF membrane

purity of GOS (%)

NA: not available. 10679



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of 3.4 × 10−8 m3 s−1 (around 2 mL min−1). Table 3 shows a comparative outlook on the purity and yield of GOS in several studies with membrane reactor that had already been proposed earlier by many researchers. It is quite evident from the table that the proposed RDMBR in this present work ultimately ends up with high yield and purity of GOS compared to its other contemporary enzymatic membrane reactor for GOS production.

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(12) Engel, L.; Schneider, P.; Ebrahimi, M.; Czermak, P. Immobilization of β-galactosidase in adsorptive membranes for the continuous production of galacto-oligosaccharides from lactose. Open Food Sci. J. 2007, 1, 17. (13) Cheryan, M. Ultrafiltration and Microfiltration Handbook; Technomic: Lancaster, 1998. (14) Grandison, A. S.; Lewis, M. J. Separation processes in the food and biotechnology industries; Woodhead: Cambridge, 1996. (15) Mountzouris, K. C.; Gilmour, S. G.; Grandison, A. S.; Rastall, R. A. Modelling of oligodextran production in an ultrafiltration stirredcell membrane reactor. Enzyme Microb. Technol. 1999, 24, 75. (16) Goulas, A. K.; Kapasakalidis, P. G.; Sinclair, H. R.; Rastall, R. A.; Grandison, A. S. Purification of oligosaccharides by nanofiltration. J. Membr. Sci. 2002, 209, 321. (17) Rice, G.; Barber, A.; O’Connor, A.; Stevens, G.; Kentish, S. Fouling of NF membranes by dairy ultrafiltration permeates. J. Membr. Sci. 2009, 330, 117. (18) Suárez, E.; Lobo, A.; Alvarez, S.; Riera, F. A.; Á lvarez, R. Demineralization of whey and milk ultrafiltration permeate by means of nanofiltration. Desalination 2009, 241, 272. (19) Bowen, W. R.; Mohammad, A. W. Diafiltration by nanofiltration: Prediction and optimization. AlChE J. 1998, 44, 1799. (20) Lipnizki, F.; Boelsmand, J.; Madsen, R. F. Concepts of industrial-scale diafiltration systems. Desalination 2002, 144, 179. (21) Bruggen, B. V.; Mänttäri, M.; Nyström, M. Drawbacks of applying nanofiltration and how to avoid them: A review. Sep. Purif. Technol. 2008, 63, 251. (22) Agenson, K. O.; Urase, T. Change in membrane performance due to organic fouling in nanofiltration (NF)/reverse osmosis (RO) applications. Sep. Purif. Technol. 2007, 55, 147. (23) Jarusutthirak, C.; Mattaraj, S.; Jiraratananon, R. Factors affecting nanofiltration performances in natural organic matter rejection and flux decline. Sep. Purif. Technol. 2007, 58, 68. (24) Juang, Y. C.; Lee, D. J.; Lai, J. Y. Fouling layer on hollow-fibre membrane in aerobic granule membrane bioreactor. J. Chin. Inst. Chem. Eng. 2008, 39, 657. (25) Moritz, T.; Benfer, S.; Á rki, P.; Tomandl, G. Influence of the surface charge on the permeate flux in the dead-end filtration with ceramic membranes. Sep. Purif. Technol. 2001, 25, 501. (26) Thanh, B. X.; Visvanathan, C.; Spérandio, M.; Aim, R. B. Fouling characterization in aerobic granulation coupled baffled membrane separation unit. J. Membr. Sci. 2008, 318, 334. (27) Nghiem, L. D.; Hawkes, S. Effects of membrane fouling on the nanofiltration of pharmaceutically active compounds (PhACs): Mechanisms and role of membrane pore size. Sep. Purif. Technol. 2007, 57, 176. (28) Splechtna, B.; Nguyen, T. H.; Steinbock, M.; Kulbe, K. D.; Lorenz, W.; Haltrich, D. Production of prebiotic galacto-oligosaccharide from lactose using β-galactosidase from Lactobacillus ruteri. Agric. Food Chem. 2006, 54, 4999. (29) Lowry, O.; Rosebrough, N. J.; Farr, A. L.; Randal, R. J. Protein Measurement with Folin Phenol Reagent. J. Biol. Chem. 1951, 193, 265. (30) Jeon, I. J.; Mantha, V. R. High performance liquid chromatography analysis of oligosaccharides formed during βgalactosidase action on lactose. J. Dairy Sci. 1985, 65, 581. (31) Das, R.; Sen, D.; Sarkar, A.; Bhattacharyya, S.; Bhattacharjee, C. A comparative study on the production of galacto-oligosaccharide from whey permeate in recycle membrane reactor and in enzymatic batch reactor. Ind. Eng. Chem. Res. 2011, 50, 806. (32) Bhattacharjee, C.; Sen, D.; Sarkar, P.; Datta, S.; Bhattacharya, P. K. Studies on the application of different ANNs to predict permeate flux in rotating disk membrane modules: A case study with MATLAB. Desalin. Water Treat. 2009, 2, 170. (33) Zemek, J.; Kuniak, L.; Gemeiner, P.; Zamocky, J.; Kucar, S. Cross-linked polyethylenimine: An enzyme carrier with spacers of various lengths introduced in cross-linking reaction. Enzyme Microb. Technol. 1982, 4, 233.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ac.in. Tel/Fax: +91 33 2414 6203. Mobile: +91 98364 02118. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work reported in this article uses the infrastructure developed under the Indo-Australian project jointly funded by DEST (Australia) and DBT (India). The Electron Microscopy Unit of Bio21 Institute, University of Melbourne, Melbourne, Victoria, Australia, is also acknowledged for their kind assistance with electron microscopy performed in the course of this research. The authors also acknowledge University Grants Commission (UGC) sponsored project [vide sanction letter no. 37-212/2009 (SR) dated January 12, 2010] and DST sponsored networking pilot program “New Indigo” on water related research with Europe (vide sanction letter no. DST/ TMC/2K11/345 dated May 17, 2012) for providing necessary infrastructure and support for carrying out this research work.

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