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Vacuum assisted membrane bioreactor for enzymatic hydrolysis of pectin from various agro-wastes

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Hydrolysis of pectins extracted from sugar beet, black currant, and red currant were studied by Aspergillus niger polygalacturonase enzyme and strong product inhibition was found. A thermostated membrane bioreactor was applied for the reaction to avoid inhibition and vacuum was used in the permeate side resulting higher product removal rate (flux) and productivity.
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Desalination 241 (2009) 29 Á33
Vacuum assisted membrane bioreactor for enzymatic
hydrolysis of pectin from various agro-wastes
K. Kiss*, N. Nemestothy, L. Gubicza, K. Belafi-Bako
Research Institute of Chemical and Process Engineering, University of Pannonia,
Egyetem u. 2., Veszprem 8200, Hungary
email: kiss@mukki.richem.hu
Received 27 August 2007; revised 9 January 2008; accepted 16 January 2008
Abstract
Hydrolysis of pectins extracted from sugar beet, black currant, and red currant were studied by Aspergillus
niger polygalacturonase enzyme and strong product inhibition was found. A thermostated membrane bioreactor
was applied for the reaction to avoid inhibition and vacuum was used in the permeate side resulting higher product
removal rate (flux) and productivity.
Keywords: Membrane bioreactors; Polygalacturonase from; Aspergillus niger ; Galacturonic acid
1. Introduction
(TMP) and/or higher ratio of membrane sur-
face/reactor volume. Higher TMP can be
Membrane bioreactors have been success-
achieved either by higher pressure in the primary
fully applied for various enzymatic reactions
side or reduced pressure (vacuum) in the
where product inhibition occurred since the
permeate side. The latter was chosen in this
membrane was able to remove continuously
work. Application of vacuum in the secondary
the product, thus inhibition could be avoided
side of the membrane bioreactor has been widely
and higher productivity was possible to achieve
applied in submerged hollow fiber bioreactors in
[1Á4]. In order to further enhance the effective-
waste water treatment [5]. However, it is hardly
ness of the product removal rate, the flux should
used in enzymatic degradation of polysacchar-
be increased, which can be realized by, for
ides and other polymers.
example,
higher
transmembrane
pressure
Pectin can be extracted from certain agro-
wastes like sugar beet pulp or press cake of berry
*Corresponding author.
fruits. It is a polysaccharide with a backbone of
Presented at the Third Membrane Science and Technology Conference of Visegrad Countries (PERMEA), Siofok,
Hungary, 2–6 September 2007.
0011-9164/09/$– See front matter # 2009 Elsevier B.V. All rights reserved.
doi: 10.1016/j.desal.0000.00.000

30
K. Kiss et al. / Desalination 241 (2009) 29 Á33
galacturonic acid linked by a-1,4 linkages [6].
boiling water [12]. The mass ratio of substanceÁ
Its derivative is D-galacturonic acid, which can
water was 1:4 and the extraction time was 4 h.
be used as a valuable raw material in the food
As a result, majority of the pectin was obtained
and pharmaceutical industry to manufacture
in the aqueous phase. Ultrafiltration was then
vitamin C or used as an acidifying agent [7].
used to clarify the extracted solution, and the
Pectic enzymes have been investigated for
diluted aqueous pectin solution was concentrated
long [8]. The apple pectin hydrolysis in free
up to 5% TSS (total soluble solid) partly by
enzyme membrane reactor (FEMR) by crude
membranes
(ultrafiltration:
polyethersulfone
enzyme (polygalacturonase and pectin lyase)
membrane, cut-off 45 kDa) and up to 30% TSS
preparation has been investigated [9,10] and
partly by evaporation. Then pectin in powder
was found that the FEMR has shown excellent
form was obtained from the concentrated pectin
catalytic activity during 15 days. Kinetics of
solution after precipitation with alcohol.
citrus pectin hydrolysis by Aspergillus niger
Shaking flask experiments were carried out in
polygalacturonase which is probably the most
a GFL 3021 (Germany) shaking incubator. The
important biocatalyst among pectinases was also
substrate solutions with various concentrations
studied [11] and strong product inhibition was
(1, 2, and 4 g/L) were prepared in citrate buffer
found to occur. In a flat sheet membrane,
(pH 4.1). To study the potential of product
bioreactor experiments were carried out for the
inhibition, galacturonic acid in different concen-
hydrolysis and productivity was enhanced. In
trations (0.5, 1, and 2 g/L) was added initially to
this work our aims were to extend the experi-
some of the reaction mixtures. Operation condi-
ments for other pectin substrates (sugar beet, red
tions were: 508C, 150 rpm, 0.01 g enzyme.
currant, and black currant), on one hand, and to
Thermostated flat sheet membrane module
achieve a further improvement in the effective-
(Fig. 1) was used as a membrane bioreactor. The
ness by applying vacuum in the permeate side
material of the ultrafiltration membrane was
and by optimizing the membrane surface/reactor
regenerated cellulose, cut-off 30 kDA (Nadir).
volume ratio, on the other hand.
Its active membrane surface area was 0.01 m2.
Reaction mixture contained the substrate and the
2. Materials and methods
Polygalacturonase enzyme from A. niger was
purchased from Sigma (USA). Its activity was
1.7 U/mg. One unit is defined as the amount of
Over
flow Feeding
Membrane
enzyme which is able to produce 1 mmol galac-
pipe vessel
module
turonic acid from polygalacturonic acid in 1 min
in pH 0/4.1 and 508C. Ethyl alcohol for pre-
Vacuum
cipitation was bought from Spektrum 3D Ltd.
Container
(Debrecen). All the other chemicals were pur-
chased from Fluka (Germany). 3-DTA (Uwa-
Equalizer
Bioreactor
Trap
tech)
test
apparatus
was
used
for
the
ultrafiltration step. The membrane material was
Magnetic stirrer
polyethersulfone (cut-off 45 kDa).
Fig. 1. Set-up of the vacuum assisted membrane bior-
Pectin substrates were extracted from sugar
eactor equipped with equalizer for continuous pectin
beet pulp, red currant, and black currant by
hydrolysis.

K. Kiss et al. / Desalination 241 (2009) 29 Á33
31
enzyme (in buffer, pH 4.1) was recirculated from
0.8
) 0.7
a reactor through the primary side of the
2 0.6
membrane module by a peristaltic pump. The
0.5
/min m
secondary side was under slight vacuum by an

0.4
0.3
ejector jet pump, controlled by a by-pass valve.
0.2
Flux (L
Permeate was collected in special traps. The
0.1
level of the substrate solution was controlled by
0
4
12
24
32
48
a mechanical equalizer.
Time (h)
The experiments in the membrane bioreactor
were carried out by using 2 g/L pectin solution
Flux with vacuum
Flux without vacuum
prepared in citrate puffer (pH 4.1). The operation
Fig. 2. Flux measured during the experiments in mem-
conditions were: 50 mL thermostated reaction
brane bioreactor.
vessel, temperature 508C, 0.01 g enzyme.
To follow the progress of the enzymatic
To avoid product inhibition the MBR seemed to
reaction, the amount of reducing sugar (galac-
be a promising solution.
turonic acid) produced was determined color-
In our previous article we presented the
imetrically with the dinitrosalicylic (DNS) acid
results of the citrus pectin hydrolysis in MBR
test [13] method using a T80 spectrophotometer
[11]. It was found that more than 40% higher
(New Century, UK).
productivity was achieved compared to the
shaking flask experiments.
3. Results
Using our new pectins as substrate the
hydrolysis was carried out in a membrane
Firstly, kinetic parameters of enzymatic
bioreactor. The set-up was modified by applying
hydrolysis of red and black currant pectins, as
vacuum in the permeate side of the membrane to
well as sugar beet pectin by A. niger polyga-
enhance flux and improve productivity.
lacturonase were determined based on shaking
The average values of flux measured during
flask experiments. The results are summarized in
the reactions are presented in Fig. 2. The flux in
Table 1, compared with our earlier data on citrus
the process using 0.25 bar vacuum was 0.7 dm3/
pectin.
min m2 (initial level) and only a slight decrease
It can be seen that considerable differences
was observed after 24 h of operation. It was
were found in the parameters and strong product
seven times higher than in our previous system.
inhibition has occurred during the hydrolysis.
Unfortunately,
this
high
flux
resulted
in
Table 1
Kinetic parameters of enzymatic hydrolysis of pectins from various sources
Pectin
Michaelis ÁMenten constant,
Maximal reaction rate,
Inhibition constant, KI (g/L)
Km (g/L)
vmax (g/L/min)
Sugar beet
1.47
0.31
1.16
Red currant
0.48
0.19
0.88
Black currant
0.79
0.31
0.94
Citrus [7]
8.30
1.06
3.13

32
K. Kiss et al. / Desalination 241 (2009) 29 Á33
14000
used earlier (without vacuum). The values of the
12000
specific productivity were 24.3, 21.5, and 19.4 g
10000
product/hg enzyme for black currant, sugar beet,
8000
and red currant pectins, respectively, compared
(mg)
6000
to the 9.7 g product/hg enzyme for citrus pectin
4000
Reducing sugar
determined earlier [7], which clearly shows the
2000
usefulness of the vacuum application.
0
0
10
20
30
40
50
60
Time (h)
Acknowledgement
Sugar beet
Black currant
Red currant
The research work was supported by GAK
Fig. 3. Amounts of polygalacturonic acid produced
(MEMBRAN5) project, grant No. OMFB-
during continuous enzymatic hydrolysis of pectins
00971/2005.
extracted from sugar beet, black and red currant.
continuously decreasing product concentrations
References
in permeate, which implies that the degradation
[1] L. Giorno and E. Drioli, Biocatalytic membrane
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18 (2000) 339 Á349.
vacuum pump was on for 10 min and off for
[2] K. Bela?-Bako, N. Nemestothy, V. Milisic and
20 min. In this way GA product concentration in
L Gubicza, Membrane bioreactor for utilisation of
the permeate was approximately constant and the
carbohydrates in waste streams, Desalination, 149
(2002) 329 Á330.
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[3] K. Bela?-Bako, Effect of operation conditions on
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the ef?ciency of membrane bioreactors, Desalina-
(accumulated) and recovered in permeate as a
tion, 200 (2006) 511 Á513.
function of time are shown in Fig. 3. As it can be
[4] A. Rektor, N. Pap, Z. Kokai, R. Szabo, Gy. Vatai
seen, it was possible to collect a slightly higher
and E. Bekassy-Molnar, Application of membrane
amount of the product in cases of black currant
?ltration methods for must processing and
and sugar beet than in the case of red currant.
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[5] D. Mourato, Water reuse with the immersed
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flux and higher effectiveness were obtained in
Enzymes in Food Processing, Academic Press,
this system than in the membrane bioreactor
New York, 1993, pp. 363 Á399.

K. Kiss et al. / Desalination 241 (2009) 29 Á33
33
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