Accepted Manuscript Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis Azis Boing Sitanggang, Anja Drews, Matthias Kraume PII: DOI: Reference:

S0960-8524(14)00842-6 http://dx.doi.org/10.1016/j.biortech.2014.05.124 BITE 13532

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 March 2014 30 May 2014 31 May 2014

Please cite this article as: Sitanggang, A.B., Drews, A., Kraume, M., Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis, Bioresource Technology (2014), doi: http:// dx.doi.org/10.1016/j.biortech.2014.05.124

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Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis

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Azis Boing Sitanggang1,3*, Anja Drews2, Matthias Kraume1

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Chair of Chemical and Process Engineering, Technische Universität Berlin. Ackerstraße 76, 13355 Berlin, Germany. 2

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HTW Berlin - University of Applied Science, Engineering II, School of Life Science Engineering. Wilhelminenhofstraße 75A, 12459 Berlin, Germany.

Department of Food Science and Technology, Bogor Agricultural University. Raya Darmaga St, Kampus IPB Darmaga Bogor 16680, West Java, Indonesia.

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*Corresponding Author:

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Name : Azis Boing Sitanggang, MSc

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Email : [email protected]

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Phone : +49-(030) 314-72693

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Fax

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Chair of Chemical and Process Engineering, Technische Universität Berlin, Ackerstraße 76, 13355 Berlin, Germany

: +49-(030) 314-72756

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Abstract

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Newly developed parallel small-scale enzymatic membrane reactors (EMRs) were used to

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enhance the synthesis of lactulose using β-galactosidase. Under batch operation, the

34

productivity of lactulose decreased abruptly from 2.72 down to 0.04 mglactulose/(Uenzymeh) over

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35 h of reaction. This was presumably caused by the action of β-galactosidase which

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performed secondary hydrolysis upon the produced lactulose. The continuous operations of an

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EMR system led to continuous removal of lactulose in the reactors restricting lactulose

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degradation caused by secondary hydrolysis. Therefore, continuous lactulose syntheses in the

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EMRs yielded significantly higher specific productivities under “steady state” conditions.

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Approximately 0.70 and 0.50 mglactulose/(Uenzymeh) for hydraulic residence times of 5 and 7 h

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were reached, respectively. Continuous lactulose synthesis performed in an EMR system

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conclusively can circumvent the drawbacks (e.g., secondary hydrolysis) of lactulose synthesis

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encountered in batch operation. It is, therefore, beneficial in terms of enhanced lactulose

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productivity and reduced enzyme consumption.

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Keywords: Enzymatic membrane reactors (EMRs), lactulose, lactose, β-galactosidase,

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transgalactosylation, process intensification.

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1. Introduction

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The potential of enzymes to catalyse bio-chemical reactions is enormous with some of the

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economically interesting products of these reactions being their intermediates rather than their

58

end-products. For these reactions, the used enzymes must have at least two functions and it is,

59

therefore, plausible that similar enzymes used to catalyse the primary reactions are also

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responsible for the degradation of the intermediates (Kirk et al., 2002). The syntheses of

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lactulose and galactooligosaccharides (GOS) lead to interesting intermediate products.

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Growing attention is recently paid to the production of lactulose and GOS as they are

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considered to be the valuable substances from the group of di-/oligosaccharides highlighting

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their prebiotic properties (Schuster-Wolff-Bühring et al., 2010). Lactulose is reported to have

65

a number of physiological effects on humans, such as enhancing colonic motility, enriching

66

the growth of probiotic bacteria, improving mineral absorptions, reducing the growth of

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putrefactive bacteria and acting as laxative agent in the treatment of constipation (Schumann,

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2002; Schuster-Wolff-Bühring et al., 2010; Seki et al., 2007).

69

There are two enzyme classes that can catalyse lactulose and GOS synthesis, such as

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glycosyltransferases and glycosidases (Hancock et al., 2006; Mayer et al., 2004). These

71

enzymes have two main functions; (i) to hydrolyse lactose and (ii) transfer the galactosyl-

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enzyme complex to galactosyl acceptors (van Rantwijk et al., 1999) (see Supplementary Data,

73

Fig. S1, modified from Shen et al., 2012). When the goal of the reaction is to hydrolyse

74

lactose using glycosyltransferases or glycosidases, water molecules act as galactosyl

75

acceptors and eventually glucose and galactose are produced as the end-products. However,

76

when other substances are used as galactosyl acceptors (i.e., fructose, alcohols etc.), a

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transgalactosylation process occurs halfway during the reactions yielding lactulose, GOS and

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other galactosides. These reactions are kinetically controlled and, therefore, undergo

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secondary hydrolysis by the actions of the same enzymes (van Rantwijk et al., 1999; Wang et

80

al., 2012). 3

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Many papers report on enzyme-catalysed lactulose and GOS syntheses (Hua et al., 2013,

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2010; Mayer et al., 2004). Most of them were carried out in batch operations with typical

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reactor working volumes of 1-500 mL (Hua et al., 2013; Martínez-Villaluenga et al., 2008;

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Mayer et al., 2004). Syntheses of these transgalactosylated products under batch operation

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normally utilise higher amounts of enzymes as the reactions have to be stopped after 8 h to

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avoid secondary hydrolysis taking place on these substances. Therefore, summarising the

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overall procedures (start-up and end activities) of lactulose and GOS syntheses it can be stated

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that batch production is presumably infeasible from an economic point of view. Many

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attempts have been made to increase the productivity of lactulose and GOS productions either

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in batch or continuous processes, including (i) the application of organic solvents as reaction

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media to reduce the amount of water in the reaction system and consequently hampering the

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hydrolysis reaction rate of the produced transgalactosylated products, (ii) the application of

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more than one enzyme during the synthesis, and (iii) other operational strategies (Hua et al.,

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2010; Schuster-Wolff-Bühring et al., 2010).

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For an alternative operational strategy, membrane reactors can be used to continuously

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remove the transgalactosylated products during the reaction. Foda and Lopez-Leiva (2000)

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reported continuous production of oligosaccharides (OS) from whey using a UF membrane

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cell operated in cross-flow filtration mode. Within their studies, a constant flux operation was

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performed by means of two peristaltic pumps. Due to the insufficient control design of the

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reactor system, the reactor could only be operated for less than 3 h at a flux value of 15

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L/(m²h). Chockchaisawasdee et al. (2005) produced GOS using β-galactosidase from

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Kluyveromyces lactis in UF membrane bioreactors. The reactors could only be operated at

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constant transmembrane pressure (TMP) and, therefore, the permeate flux declined over the

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reaction course as cake layer deposition or fouling occurred on top of the UF membranes.

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Thereby, the hydraulic retention time (HRT) increased during the process which led to rising

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concentrations of GOS inside the reactor over time. As a consequence, secondary hydrolysis 4

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of the produced GOS occurred faster. Considering these conditions, a proper control design

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for a constant flux (i.e., HRT) operation during GOS and other transgalactosylation products

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is of importance to restrict the impact of secondary hydrolysis taking place.

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Mayer et al. (2010) synthesised lactulose in an EMR and in a packed-bed reactor (PBR)

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continuously using β-glycosidase from Pyrococcus furious. The lactulose production in the

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EMR was unsuccessful because the half-life of the enzyme was less than a day and further

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investigations were not reported. On the other hand, the synthesis of lactulose in a PBR was

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successful using the immobilised β-glycosidase. It is, however, assumed to be quite tedious as

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the reported preparation of the enzyme immobilisation took more than 24 h.

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Considering the economic value of the positive physiological properties of lactulose, there is a

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need to develop a proper reactor and control system for lactulose synthesis to avoid secondary

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hydrolysis occurring on the produced lactulose during the reaction. Hence, this study aimed to

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further develop a screening and characterisation system based on parallel small-scale EMRs

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(Lyagin et al., 2010) which allows continuous production of lactulose at a constant flux

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operation with a short HRT. The two reactors in parallel make it possible to obtain duplicates

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and reproducible results in a shorter space of time. Developing a control design for the reactor

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system was firstly carried out to enable a constant flux operation (thus constant HRT) for a

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long period of reaction. Moreover, continuous lactulose syntheses were conducted at different

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HRT values and their productivities were compared with the one from the synthesis of

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lactulose in a batch process.

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2. Material and methods 2.1 Material 2.1.1 Reactor configuration

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Initially, two parallel EMRs were built for batch and continuous operation purposes. The

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scheme of the developed EMR system is shown in Fig. 1. An EMR consisted of a pressure-

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stable glass container and a body (holder) which was modified from the XFUF-047 dead-end 5

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test cell, Merck Millipore Darmstadt, Germany. These modifications were necessary to insert

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the temperature, pH and substrate probes inside the reactor (see Fig. 1). The maximum

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working volume of the reactor was 90 mL with a flat-sheet PES membrane (effective surface

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area = 12.38 x 10-4 m²) placed at the bottom of the reactor. Piping (PTFE ID = 0.8 mm; OD =

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1.6 mm) and other small parts for fittings were received from VWR International GmbH, and

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Th. Geyer Berlin GmbH, Germany. UF membranes made of polyethersulfone (PES) with a

140

molecular weight cut-off (MWCO) of 10 kDa were obtained from Microdyn-Nadir GmbH,

141

Germany. The pressure-stable pH electrode (glass, Pmax = 6 bar, Tmax = 130 oC, 8 x 120 mm)

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was purchased from ProSense BV, The Netherlands. For permeate measurement, a Kern EW

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620-3NM precision balance was employed (Kern & Sohn GmbH, Germany). In addition, the

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proportional pressure regulator MPPE (Festo AG & Co. KG, Germany) was used for

145

continuous operation.

146

Data acquisition (DAq) was realised as a combination of several National Instruments (NI)

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modules, such as (i) NI 9201 16-channel, 100 kS/s/ch, 16-Bit, ±10 V analogue input module;

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(ii) NI 9870 4-port, RS232 serial interface module and (iii) NI 9264 16-channel analogue

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output module. These modules were mounted on a cRIO-9076 integrated 400 MHz real-time

150

controller and LX45 FPGA chassis system produced by National Instruments, Germany. This

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typical chassis can fully control up to 12 parallel reactors, enabling the feasibility of this

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system as a rapid screening and characterisation system. The software Laboratory Virtual

153

Instrument Engineering Workbench (LabVIEW) Professional, version 2012 was employed to

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control the reactors and save all data.

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2.1.2 Chemicals

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The enzyme β-galactosidase from Kluyveromyces lactis (EC Number 232-864-1, G3665),

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acetonitrile (271004), 2-Nitrophenyl β-D-galactopyranoside (ONPG, 73660), 2-Nitrophenol

158

(ONP, 19702), lactulose (61360), D-fructose (F0127), lactose (17814) were purchased from

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Sigma-Aldrich, Germany. All other chemicals were analytical grade, obtained from Merck-

160

Millipore and from VWR International GmbH, Germany.

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2.2 Control design and strategy for constant flux operation

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Permeate mass was quantified by means of a precision balance and converted into volume for

164

calculating its flow rate. Furthermore, the flow rate served as an actuator for controlling flux

165

or HRT. With regards to the controller output (CO; pressure, bar), and process variable (PV;

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flux, L/(m²h)), the filtration process showed a non-periodic response that follows a PT1T0

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model as process dynamics (Schwarze, 1962) and has been reported elsewhere (Lyagin et al.,

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2010). These process dynamics were generally recognised as first order plus dead time

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(FOPDT) behaviour. To control such process dynamics, a proportional-integral-derivative

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(PID) controller was implemented in the EMR system. Moreover, PID parameters were tuned

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according to the method described by Kuhn (1995).

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The flux (and thus also HRT) was feed-back controlled in the continuous process (see Fig. 1).

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When the setting value (SV) of flux was inserted into the LabVIEW program, the program

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automatically sent an analogue input commanding the proportional pressure regulator (3) to

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open its valve allowing a certain amount of gas to be released from the N2 bottle (1). As the

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substrate tank (4) was pressurised, substrate solution was fed into the reactor (5). Since the

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reactor was completely filled with liquid, the additional volume from the substrate tank

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consequently drove the same amount of liquid out of the reactor via the membrane which was

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then collected by the precision balance (8). In the LabVIEW program this permeate mass was

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converted to its volume (using the corresponding density of the solution) and eventually into

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the real flux value (process variable, PV) using the membrane effective surface area and the

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time of filtration. This loop was carried out within one second (1 s). Furthermore, the

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difference between PV and SV was used by the PID controller to give the subsequent

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command to the proportional pressure regulator again (3). This cycle was repeated during the

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whole reaction process, giving the possibility to control a flux and thus HRT precisely.

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2.3 Enzyme filtration

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In order to study the membrane performance, enzyme filtration was conducted continuously

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at an HRT of 3 h for 25 h. The significant difference between the membrane MWCO (i.e.,

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10 kDa) and the enzyme’s molecular weight (i.e., β-galactosidase ~ 465 kDa (Appel et al.,

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1965; Juers et al., 2000)) should give a complete rejection of the enzyme molecules. By

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quantifying the protein concentration in the permeate (see section 2.6.2), the membrane

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rejection was found to be 95 %. As reported by Lozano et al. (2014), a PES membrane with

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10 kDa MWCO could also reject 99 % of a cellulase complex (celluclast 1.5L®) and 98 % of

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cellobiase (novozym 188).

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2.4 Operational procedure for lactulose synthesis

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Lactulose was synthesised in the developed EMR system run respectively in batch and

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continuous mode, using a bi-substrate (lactose and fructose) and K. lactis β-galactosidase. In

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contrast to enzymes from the group of glycosyltransferases that usually need activated

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substrates and cofactors, β-galactosidase (group of glycosidases) is generally cofactor

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independent (Mayer et al., 2004). In addition, it is more relevant for industrial applications

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(e.g., for hydrolysis of lactose) as it is commercially available and relatively inexpensive

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(Mayer et al., 2004; Perini et al., 2013).

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Under batch operation, a series of reactions was carried out without enzyme removal from the

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reactor. A 1.0 mL sample was taken from the permeate side and compensated by the same

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amount (i.e., 1.0 mL) of fresh substrate conveyed into the reactor at 0.5; 2: 3; 5; 8; 10 and 12

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h. In order to enable this sampling procedure, a pressure (i.e., 0.5 bar) was applied in the 8

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substrate tank manually. Though an exchange between inlet and permeate took place in the

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system, the operation was still considered as a batch reaction as it had a very long HRT.

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The effects of several parameters, such as total sugar concentration (g/L), enzyme

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concentration (U) and molar ratio of lactose to fructose (mL/mF) on the lactulose production

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were investigated in batch operation. For continuous operation, the concentration of lactulose

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was monitored for different HRT values, initially 5 and 7 h. In both types of operation (batch

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and continuous), the reactors were fully filled with a working volume of 90 mL, 50 mM

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buffer phosphate pH 6.8 as the reaction medium, stirred at 200 rpm and incubated at a

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constant temperature of 40 ± 1 °C.

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The productivities of batch and continuous processes (at different HRT values) were

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compared in terms of specific productivity calculated as follows:

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(1)

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where Ci = lactulose concentration at a certain sampling time ti, Vi = permeate volume

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collected until ti (for batch mode Vi = 90 mL), ti = sampling time.

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2.6 Analysis 2.6.1 Measurement of the enzyme activity

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ONPG was used as a substrate to determine the activity of β-galactosidase. The procedure

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was similar to the method reported by Hua et al. (2010) with several minor modifications. The

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enzyme β-galactosidase generally hydrolyses ONPG to yield ONP. The color of the ONP

229

solution is yellow and can be monitored at 420 nm using a spectrophotometer (Specord ® 210

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Plus – Analytik Jena AG, Germany). One unit (1 U) of enzyme activity is defined as the

231

amount of enzyme required for liberating the equivalent 1 µmol ONP per minute at 30 oC and

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pH 6.8.

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2.6.2 Determination of mono-, disaccharides and protein concentration

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For mono- and disaccharide analysis, an HPLC was used, equipped with a Vertex Plus 250 x

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4.6 mm Eurospher II 100-3 NH2 column (Knauer GmbH, Germany), a WellChrom K-2300 RI

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detector and a K.1001 pump combined with an electric valve drive. The evaluation of the

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resulting chromatograms was done by the software Eurochrom 2000. The mobile phase was a

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mixture of acetonitrile and water (75:25) with an isocratic gradient, pumped at a constant flow

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rate of 1.0 mL/min. Column temperature was set to 30 oC. For achieving a proper resolution,

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samples were diluted with ultra-pure water for four times and with the mobile phase at a ratio

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of 1:1 prior to injection. Finally 20 µL of the diluted sample were thoroughly injected into the

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HPLC. The retention time was 30 min.

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The enzyme concentration (expressed as protein concentration) in the permeate during the

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enzyme filtration test was determined using the linearised Bradford method (Zor and Selinger,

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1996). The protein concentrations (0-0.4 g/L) and their absorbance ratios at 590 nm and

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450 nm were plotted to give a linear line.

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3. Results and discussion 3.1 Flux stability

252

Tuning PID controller parameters (P, I and D) is still one of the most highlighted topics in

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control design and it sometimes becomes the bottleneck of PID controller’s wider applications

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(Skogestad, 2003). Tuning PID controllers basically depends on the process dynamics of the

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plant. Therefore, systematic procedures to find suitable values for those parameters are

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required (Skogestad, 2003). There are many strategies that have been introduced for tuning

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PID parameters and have been extensively summarised in several works by different authors

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(Aidan, 2009; Madhuranthakam et al., 2008; Syrcos and Kookos, 2005). Out of those, a

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strategy introduced by Kuhn (1995) was selected within this study which also had been used

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elsewhere (Lyagin et al., 2010). This strategy is based on an open-loop approach which is 10

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considered not to be as time-consuming as the closed-loop one, because there is no need to

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wait for several periods of oscillations during several trial-and-error attempts. Within this

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study, the values for PID parameters were 0.0075, 0.360 min, and 0.087 min, respectively for

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P, I and D. This setting was tested in the filtration of lactose and fructose with a total sugar

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concentration of 500 g/L in a serial flux stepping operation. As can be seen in Fig. 2, when

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the SV shifted, the CO changed accordingly to bring the PV as close as possible to the SV.

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The CO was not jerky and consequently the PV responses were stable without producing any

268

overshoot response. The error between SV and PV was evaluated to be less than 5 %. This

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result was comparable to the study of tuning PI/D parameters in a closed-loop method

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reported by Skogestad (2003) and Shamsuzzoha and Skogestad (2010).

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3.2 Transgalactosylation towards lactulose 3.2.1 Effects of the total sugar concentration (g/L), enzyme concentration (U) and molar ratio of lactose to fructose (mL/mF)

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The investigation of the effects of several parameters (sugar concentration (g/L), enzyme

276

concentration (U) and molar ratio of lactose to fructose (mL/mF)) on the productions of

277

lactulose was done in batch processes. As can be seen in Fig. 3, a higher sugar concentration

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constantly yielded a higher lactulose production. The maximum concentrations of lactulose

279

obtained were 6.69; 8.24 and 11.0 g/L for sugar concentrations of 300, 400 and 500 g/L,

280

respectively. A smaller amount of lactulose for a lower sugar concentration (i.e., 300 g/L) was

281

caused by a lower availability of the galactosyl-enzyme complex which was necessary to react

282

with fructose. In relation to these results, the yields of lactulose also increased slightly from

283

0.03 to 0.031 and 0.034 glactulose/glactose for sugar concentrations of 300, 400 and 500 g/L,

284

respectively. Lee et al. (2004) also reported an increased level of lactulose concentration at a

285

higher concentration of the substrate (a mixture of lactose and fructose) at a constant molar

286

ratio of lactose to fructose (mL/mF).

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The small amount of enzyme (i.e., 100 U) was assumed to be inhibited in the highly

288

concentrated substrate since higher lactulose production was obtained when the enzyme

289

concentration was brought up to 300 U (Fig. 4a). However, at level of 500 U of enzyme

290

concentration, the obtained lactulose concentration was not much altered. Similar results have

291

been reported by Kim and Oh (2012) where the concentration of lactulose increased by the

292

increase of the enzyme load and eventually reached the maximum lactulose concentration at

293

optimum enzyme load (150 U/mL). In Fig. 4a, lactulose concentrations were up to 14.1 and

294

13.76 g/L for 300 and 500 U, respectively. Within this study, an enzyme concentration of 300

295

U was considered to be the nearly optimum enzyme concentration. When the enzyme

296

concentration was 500 U, the lactulose concentration reduced as compared to that of 300 U.

297

Excessive amount of enzyme was unfavorable since it led to the acceleration of both primary

298

and secondary hydrolysis (see Fig. 4a) and eventually resulted in a lower lactulose

299

concentration and higher lactose consumption.

300

It is worth mentioning that higher enzyme concentrations performed the transgalactosylation

301

faster. In the course of the reaction at 100 U of enzyme concentration, the maximum lactose

302

concentration (i.e., 11.0 g/L) was reached after 23 h (data not shown) whereas for 300 and

303

500 U the optimum reaction times were 5 and 3 h, respectively. Secondary hydrolysis

304

generally occurred as soon as lactulose was produced. This was due to secondary hydrolysis

305

being a kinetically controlled reaction. Moreover, secondary hydrolysis appeared more

306

visible, after the maximum concentration of lactulose has been obtained. As an example, at

307

300 U and after 5 h reaction, the lactulose concentration gradually decreased to 13.55, 12.36

308

and 11.60 g/L for 8, 10 and 12 h, respectively. Highlighting on this issue which typically

309

occurs in batch operation, a strategy for continuous removal of lactulose is of importance.

310

This withdrawal consequently needs a sufficient control design to control flux precisely

311

during the reaction. Based on lactose consumption (see Fig. 4a), it could be stated that the

312

influence of β-galactosidase on the primary hydrolysis (i.e., that of lactose) was not affected 12

313

by the lactulose secondary hydrolysis. Lactose consumption increased over the reaction time

314

to 95-98 % within 12 h. Formation of by-products, which were confirmed as GOS by Shen et

315

al. (2012), was also observed during the reaction. This was indicated by the increased number

316

of peaks that appeared in the HPLC chromatograms (data not shown). Interestingly,

317

secondary hydrolysis also occurred on those GOS. This was reflected by the reduction of the

318

peak areas of the GOS in the HPLC chromatograms after the maximum concentrations of

319

transgalactosylated products (lactulose, GOS) reached (data not shown).

320

Increasing the mL/mF ratio was found to decrease the produced lactulose concentration (Fig.

321

4b). When the mL/mF ratio was brought to 0.5, a lactulose concentration of 16.70 g/L was

322

obtained, whereas for a ratio of 2.0 and 1.0, lactulose concentrations were 8.64 g/L and 14.10

323

g/L, respectively, at 5 h reaction time. At a lower mL/mF ratio (i.e., 0.25), lactulose

324

concentration was enhanced to 17.91 g/L at 8 h. The yield of lactulose concentration reported

325

by Lee et al. (2004) was about 5 % (based on initial lactose concentration) or equal to19.6 g/L

326

using 10.4 g/L permeabilised cells of Kluyveromyces lactis at an optimum mL/mF ratio of 1.0.

327

Mayer et al. (2004) reported a maximum lactulose concentration of 16 g/L using β-

328

glycosidase from P. furiosus (CelB) at an optimum mL/mF ratio of 1/15. Apart from the types

329

of enzymes used during the synthesis of lactulose, lactulose concentration is strongly

330

dependent on mL/mF ratio (Guerrero et al., 2011). A large amount of fructose seems to be

331

advantageous for lactulose synthesis, as this composition allows a higher probability for

332

fructose to react with the galactosyl-enzyme complex (Guerrero et al., 2011; Hua et al., 2010).

333

It eventually can overshoot the transgalactosylation, resulting in a higher lactulose production.

334

However, it must be noted that higher amounts of fructose can also inhibit the catalytic

335

activity of the enzyme (i.e., β-glycosidase) as reported by Mayer et al. (2004). For further

336

studies in continuous lactulose syntheses, 500 of g/L sugar concentration, an enzyme

337

concentration of 300 U and an mL/mF ratio of 0.5 were used in the reactions.

13

338

3.2.2 Continuous syntheses of lactulose at different HRTs

339

Continuous syntheses of lactulose at two different HRT values were conducted for 35 h. As

340

can be seen in Fig. 5a, the reaction reached highest concentrations at 10 and 12 h,

341

respectively, at HRTs of 5 and 7 h. The maximum lactulose concentrations obtained for HRTs

342

of 5 and 7 h were almost similar, approximately 12 g/L. Nevertheless, it must be noted that

343

lactulose concentration at longer HRT (7 h) was slightly higher (~ 2 %) than at the shorter one

344

(5 h).

345

In batch operation (conditions: [sugar] = 500 g/L, [E] = 300 U, mL/mF ratio = 0.5), the

346

maximum concentration of lactulose was 16.70 g/L at 5 h. However, lactulose concentration

347

reduced remarkably down to 4.87 g/L over 35 h (Fig. 5a). This was presumably caused by the

348

action of β-galactosidase which performed secondary hydrolysis during the batch process.

349

Besides the sharp reduction of lactulose concentration, the primary hydrolysis also took place

350

rapidly in the batch operation leading to a lower level of the remaining lactose in the reactor

351

(down to 2.0 % after 35 h reaction, see also section 3.2.1). In comparison, the remaining

352

lactose concentrations in the reactor during continuous operations at different HRTs were

353

higher than in batch operation. The remaining lactose concentrations at 5 and 7 h HRT were

354

11.70 and 10.60 %, respectively.

355

Although at nearly constant lactulose concentration the remaining lactose concentrations at

356

the two HRT values were not significantly different, Pspec at 5 h HRT was vividly higher than

357

at 7 h, since with a shorter HRT a higher amount of permeate was produced over the time.

358

The values were 0.70 and 0.50 mglactulose/(Uenzymeh), respectively, for HRTs of 5 and 7 h over

359

35 h (Fig. 5b). Pspec in the batch process at 0.5 h was four times higher than the maximum Pspec

360

in continuous operation at HRT of 5 h. This might lead to the conclusion that batch operation

361

was superior to continuous ones and, therefore, that economically batch would be preferred.

362

However, when the unproductive time for start-up and end activities (preparation, filling,

363

heating, cleaning etc.) which was evaluated to be around 3.5 h, was considered in every cycle 14

364

of batch operation, its maximum Pspec was nearly similar to that continuous operation at 5 h

365

HRT, around 0.70 mglactulose/(Uenzymeh) (see the curve of normalised batch in Fig. 5b). The

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advantage of continuous operation at 5 h HRT was even more, as this maximum value of Pspec

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(0.70 mglactulose/(Uenzymeh)) remained nearly constant for 35 h and it was not encountered in

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batch operation.

369

The selectivity values obtained from batch operation and continuous operations at different

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HRT values (5 and 7 h) were also evaluated (Fig. 5c). Selectivity is defined as the ratio

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between the amount of produced lactulose (mol) and consumed lactose (mol). The maximum

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selectivity achieved by batch operation was slightly higher than the continuous ones, up to

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0.07 mollactulose/molcons. lactose at 5 h. For continuous syntheses (5 and 7 h HRT), the selectivity

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values were almost similar, approximately 0.055 mollactulose/molcons. lactose at 10 h. As

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mentioned previously (see also Fig. 5a), the remaining lactose concentrations for both

376

continuous syntheses remained nearly constant after 12 h (thus lactose consumptions were

377

constant), whereas for batch operation the remaining lactose concentration reduced

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remarkably till 1 % within 35 h. Hereby, the syntheses of lactulose under 5 and 7 h HRT were

379

also propitious because the reaction selectivity values only slightly decayed (2-2.5 % of

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reduction) during 35 h reaction whilst the selectivity in batch operation reduced drastically

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down to 0.020 mollactulose/molcons. lactose (72 % of reduction).

382

It is obvious that continuous synthesis of lactulose acquired better production performance

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than the batch process in terms of Pspec. This was due to the capability of the continuous

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process to maintain “steady state” conditions with only a slight reduction of the lactulose

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concentrations (app. 3-4 % of reduction, see Fig. 5a) over 35 h of reaction course and the fact

386

that the produced lactulose was continuously withdrawn from the reactors insofar the

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reaction. The reduction of lactulose concentration was presumably caused by enzyme

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inactivation. Factors that might contribute to the inactivation were thermal inactivation, shear-

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induced inactivation and adsorption of enzyme molecules on top of the membrane (Ganesh et 15

390

al., 2000; Thomas and Geer, 2011). Conclusively, continuous synthesis of lactulose in an

391

EMR system can circumvent secondary hydrolysis which is the main obstacle in batch

392

operation and thus maintain the selectivity of the reaction.

393

To our knowledge, there is only one published work about continuous synthesis of lactulose

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in an EMR using β-glycosidase from Pyrococcus furiosus (CelB). Mayer et al. (2010)

395

reported the synthesis of lactulose to be stable for less than one day in an EMR. Within their

396

studies, a less stable continuous lactulose production in an EMR was thought to be influenced

397

by the small mL/mF ratio (i.e., 1/15) and an unspecific enzyme adsorption onto the membrane

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at initial operation of the reactor. Though in their previous studies (Mayer et al., 2004) such

399

an optimum mL/mF ratio of 1/15 had been chosen, this ratio was still considered to have an

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influence on the reduction of β-glycosidase activity in a long period of operation (continuous

401

process). Within this study, after 35 h, the synthesis of lactulose using this EMR system was

402

still at steady-state. Further studies of lactulose syntheses are still on-going using the

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developed EMR system for a long-term operation (i.e., ~100 h).

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Besides the enhanced productivity of lactulose and the reduced enzyme consumption in

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continuous lactulose synthesis, there is still a major issue that has to be pointed out when

406

operating an EMR system. As a consequence of the constant flux operation, the TMP

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increases over the reaction time due to the cake deposition or fouling. The increase of TMP

408

was higher at an HRT of 5 h than at 7 h (Fig. 6). Higher permeate flux resulting from a shorter

409

HRT leads to a higher rate of material (foulant) to be deposited onto the PES membrane.

410

Similar results have been reported elsewhere, and enzyme molecules are considered to

411

contribute to such fouling phenomena (Luo et al., 2013). From Fig. 6, it is, however, seen that

412

the differences between initial and final TMP for both HRT values were not so significant.

413

Therefore, a longer continuous lactulose synthesis using this EMR system is highly possible

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regarding the operational time span of the membrane.

415 16

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3.2.3 Performances of EMRs – precision of parallel reactors

418

For an efficient and fast process design, parallel reactors are normally used. The difference

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between the performances of the reactors in the EMR system was therefore to be considered.

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For both HRTs (5 and 7 h), the similarity of the reactors (evaluating those two reactors)

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during continuous lactulose productions was at a high level, about 95 %. As mentioned

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earlier, the PID controller embedded within this EMR system could monitor up to 12 parallel

423

reactors. The developed EMR system (in this study) holds prospective possibilities to be

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expanded (increasing the number of the reactors) without affecting the precision of each

425

reactor. Such a parallel system can circumvent the well-known bottleneck during bioprocess

426

development, namely the step from batch-wise lab-scale studies to continuous full-scale

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operation. Such long-term continuous studies are needed to initially evaluate optimum

428

reaction conditions prior to scaling up the EMR system.

429 430

4. Conclusions

431

A newly developed EMR system supported by a suitable control design was used to enhance

432

lactulose synthesis. Under batch operation, lactulose production was significantly dependent

433

on mL/mF ratio. In continuous operation, a shorter HRT (5 h) yielded a higher specific

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productivity compared to the longer one (7 h). The secondary hydrolysis could be

435

circumvented during continuous operation, indicated by nearly constant lactulose

436

concentration lasting for up to 35 h. Optimisation of reaction conditions, such as HRTs,

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agitation speeds, ionic strength of the media, etc. can possibly further enhance continuous

438

synthesis of lactulose using this developed EMR system.

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441 442

Acknowledgement

443

German Academic Exchange Service (DAAD) is gratefully acknowledged for providing a

444

PhD scholarship to the first author. In addition, the authors would like to thank Gabriele

445

Hedicke and Bernd Schmidt for the technical assistances given during this study.

446 447

References

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List of Figures

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Fig. 1. Schematic of the developed EMR system; (1) N2 bottle, (2) pressure reducer, (3) proportional pressure regulator, (4) substrate tank, (5) reactor, (6) flat-sheet PES membrane, (7) heating system, (8) precision balance. Q = quality parameter, pH. Dashed lines (- - -) indicate control lines.

550 551 552

Fig. 2. Process variables with their corresponding pressures as controlled by PID controller in a series of setting values (30, 60, 50 and 75 L/(m2h)). Solution used was a mixture between lactose and fructose at level of 500 g/L.

553 554 555

Fig. 3. Effect of total sugar concentration on the production of lactulose using β-galactosidase from Kluyveromyces lactis, [E] = 100 U, mL/mF ratio = 1.0, phosphate buffer pH 6.8, stirred at 200 rpm, 40 °C. (a) [sugar concentration] = 300, (b) 400, (c) 500 g/L.

556 557 558 559 560 561 562 563

Fig. 4. Effects of (a) enzyme concentration [E] (mL/mF ratio = 1.0) and (b) mL/mF ratio ([E] = 300 U) on the productions of lactulose using β-galactosidase from Kluyveromyces lactis, [sugar concentration] = 500 g/L, phosphate buffer pH 6.8, stirred at 200 rpm, 40 °C.

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Fig. 6. Flux profiles and TMP evolutions during continuous syntheses of lactulose at different HRTs.

Fig.5. (a) Profiles of lactulose concentrations, (b) Pspec. and (c) selectivity values of batch and continuous lactulose productions using β-galactosidase from Kluyveromyces lactis, [sugar concentration] = 500 g/L, [E] = 300 U, mL/mF ratio = 0.5, phosphate buffer pH 6.8, stirred at 200 rpm, 40 °C.

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21

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Continuous synthesis of lactulose in an enzymatic membrane reactor reduces lactulose secondary hydrolysis

569

Azis Boing Sitanggang1,3*, Anja Drews2, Matthias Kraume1

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1

Chair of Chemical and Process Engineering, Technische Universität Berlin. Ackerstraße 76, 13355 Berlin, Germany. 2

573 574 575 576

3

HTW Berlin - University of Applied Science, Engineering II, School of Life Science Engineering. Wilhelminenhofstraße 75A, 12459 Berlin, Germany.

Department of Food Science and Technology, Bogor Agricultural University. Raya Darmaga St, Kampus IPB Darmaga Bogor 16680, West Java, Indonesia.

577 578 579 580 581 582 583

*Corresponding Author:

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Name : Azis Boing Sitanggang, MSc

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Email : [email protected]

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Phone : +49-(030) 314-72693

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Fax

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Chair of Chemical and Process Engineering, Technische Universität Berlin, Ackerstraße 76, 13355 Berlin, Germany

: +49-(030) 314-72756

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Highlights

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Continuous operation was carried out at precisely controlled flux (i.e., HRT).

600


Secondary hydrolysis decreased by removal of lactulose during reaction.

601


Enhanced productivity of lactulose synthesis was achieved in continuous operation.

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An efficient reduction of enzyme consumption in continuous synthesis was realised.

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Continuous lactulose production for ~ 100 h is amenable.

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23