Continuous removal of hydrogen peroxide with

Water Science & Technology Vol 55 No 1–2 pp 27–33 Q IWA Publishing 2007
immobilised catalase for wastewater reuse
D.S. Yoon*,**, K. Won*†, Y.H. Kim*,***, B.K. Song*, S.J. Kim****, S.-J. Moon* and B.S. Kim**
*Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong-gu, Daejeon 305-343, Korea
(E-mail: kwon@krict.re.kr)
**Department of Chemical Engineering, Chungbuk National University, 12 Gaesin-dong, Heungduk-gu,
Cheongju 361-763, Korea
***Department of Chemical Engineering, Kwangwoon University, 447-1 Wolgye-dong, Nowon-gu, Seoul
139-701, Korea
****Green and Global EnviTech Co., 39-3 Seongbok-dong, Yongin 449-795, Korea

Abstract Hydrogen peroxide was continuously removed for wastewater reuse using an immobilised
biocatalyst. A commercial catalase, which is an enzyme to decompose hydrogen peroxide to water and
oxygen, was entrapped in chitosan beads. Hydrogen peroxide in aqueous solutions of varying pH,
temperature and concentration was continuously removed through a reactor containing the catalase-
entrapped chitosan beads at high efficiency for 24 h. Additional silicate coating of the chitosan beads
resulted in significant improvements in the catalase performance under harsh conditions, which are often
found in peroxide-based industrial processes. We expect that immobilisation of catalases can enhance their
applicability for continuous degradation of hydrogen peroxide for wastewater reuse.
Keywords Catalase; chitosan; hydrogen peroxide; immobilisation; silicate coating; wastewater reuse

Introduction
Hydrogen peroxide (H2O2) is a powerful oxidant which is widely used as an alternative
to toxic chlorine (Lin and Gurol, 1996; Charron et al., 2004). Hydrogen peroxide is
much less hazardous than chlorine, but there is still a need for better ways to remove
H2O2 so process water can be reused or returned to the environment. Currently, hydro-
gen peroxide is either diluted with large quantities of clean water, which is expensive
and wasteful, or treated with agents like sodium bisulphite or hydrosulphite, which
leaves salt in the water (Borman, 2003). Alternatively, the application of catalases has
been suggested. A catalase is an abundant enzyme in nature that promotes the conver-
sion of hydrogen peroxide into water and oxygen. Catalases have been used for elimin-
ation of residual hydrogen peroxide in textile, food and semiconductor industries
(Tarhan, 1995; Costa et al., 2002; Oh et al., 2002). However, the high cost of the
enzyme has hampered its application, even though a catalase is one of the most effec-
tive enzymes. To reuse enzymes through immobilisation can reduce the cost. Enzyme
immobilisation also prevents catalases from flowing out and interacting with the next
process (Fruhwirth et al., 2002). Catalases have been immobilised in various organic/
inorganic supports (Betancor et al., 2003; Vera-Avila et al., 2004). Chitosan, a polymer
of non-acetylated (1 ! 4)-b-linked D-glucosamine residues, is inexpensive, hydrophilic,
biocompatible and thus attractive for enzyme immobilisation (Krajewska, 2004). A com-
mercial catalase was immobilised on chitosan film (Çetinus and Öztop, 2000) and into

†
Department of Chemical and Biochemical Engineering, Dongguk University, 3-26 Pil-dong, Chung-gu, Seoul 100-715,
Korea (E-mail: keehoon@dongguk.edu)

doi: 10.2166/wst.2007.016 27
 chitosan beads (Çetinus and Öztop, 2003). In this work, we have entrapped a commer-
cial catalase in chitosan beads and carried out continuous removal of H2O2 for waste-
water reuse using the catalase-entrapped chitosan beads. The effect of pH, temperature
and H2O2 concentration on the removal efficiency has been examined. In order to
improve the performance under harsh conditions, such as low pH, high temperature and
high H2O2 concentration, silicate coating of the chitosan beads has also been conducted
in the present study.
D.S. Yoon et al.

Materials and methods

Materials
A commercial catalase (Terminoxe Ultra 50L) was provided by Novozymes, Denmark
and used as received. According to the supplier, Terminox Ultra is a yellowish to light
brown liquid containing a catalase produced by submerged fermentation of a genetically
modified Aspergillus microorganism. A hydrogen peroxide solution (30%) was purchased
from Junsei, Japan. Chitosan (medium molecular weight), sodium tripolyphosphate, and
tetramethyl orthosilicate (TMOS) were obtained from Aldrich, USA.

Entrapment of catalase in chitosan beads

A chitosan solution, which was prepared by adding 3 g of chitosan to 100 ml of acetic acid
solution (1% v/v), was mixed with 2 mL of Terminox Ultra. Spherical catalase-entrapped
chitosan beads were formed by dripping the mixture into 500 mL of tripolyphosphate
(TPP) solution (3% w/v) with a syringe pump. They were left overnight for hardening, and
then were washed on a filter two times with distilled water. The filtered TPP solution and
the two washings were collected for entrapment efficiency determination.
 
Ci V i 2 Cf V f
Entrapment efficiency ð%Þ ¼ £ 100 ð1Þ
Ci V i

where Ci is the initial protein concentration, Vi is the initial volume of enzyme solution, Cf
is the protein concentration in the total filtrate and Vf is the total volume of the filtrate.

Coating of the beads with silicate

The catalase-entrapped chitosan beads (about 50 g) were placed in hexane (300 mL) to
cover the beads. Seventy millilitres of TMOS was added and the mixture was left over-
night at room temperature to complete the polymerisation process. Finally, the beads
were filtered from the solution and then washed with distilled water.

Continuous H2O2 removal using immobilised catalase

The catalase-entrapped chitosan beads with or without silicate coating were packed into a
water-jacketed column. For continuous H2O2 removal, a hydrogen peroxide solution was
continuously pumped into the reactor at 3 mL/min. Concentration, pH and temperature of
H2O2 solution were varied from 10 to 100 mM, from 2 to 10, and from 20 to 90 8C,
respectively. H2O2 contents in the effluent from the reactor were monitored by measuring
the absorbance at 240 nm for 24 h.

Determination of protein concentration and catalase activity

Protein concentrations were measured with the BCA Protein Assay Reagent Kit (Pierce,
USA) using a standard protocol. Specific activity of free and immobilised catalase is
defined as activity per mg protein free and entrapped in the beads, respectively. The cata-
28 lytic activity was determined by measuring the production rate of oxygen formed by the
reaction using a digital flowmeter (DFC-HRe, Alltech, USA). The reaction was carried
out in 300 mL of sodium phosphate buffer (0.1 M, pH 7.2) containing 10 mM H2O2 at
20 8C. The amount of protein in the beads was determined from the entrapment efficiency
shown in equation (1).
(a) 100

D.S. Yoon et al.

99
H2O2 removal efficiency

98

97

pH 4.0
96 pH 7.2
pH 10.0

95
0 5 10 15 20 25 30
Time (h)
(b) 100

99
H2O2 removal efficiency

98

97
Temp. 20oC
Temp. 40oC
96
Temp. 60oC
Temp. 70oC

95
0 5 10 15 20 25 30
Time (h)

(c) 100

99
H2O2 removal efficiency

98

97

10mM H2O2
96 30mM H2O2
50mM H2O2
95
0 5 10 15 20 25 30
Time (h)

Figure 1 Continuous removal of H2O2 using the catalase-entrapped chitosan beads. (a) pH effect at 20 8C
and 10 mM; (b) temperature effect at pH 7.2 and 10 mM; (c) H2O2 concentration effect at pH 7.2 and
20 8C 29
 Morphology and elemental analysis of the beads
The surface of non-coated and silicate-coated chitosan beads was observed using an
environmental scanning electron microscope (ESEM; Quanta 400, FEI, USA), which
enabled us to view specimens in their natural states. Elemental analysis of the beads
in ESEM was performed with an energy dispersive spectrometer (EDS; SuperDry II,
Noran).
D.S. Yoon et al.

Results and discussion

Continuous H2O2 removal using catalase-entrapped chitosan beads
For continuous removal of hydrogen peroxide, a commercial catalase was immobilised
in chitosan beads. Chitosan, a polycationic polysaccharide derived from the natural
polymer chitin, forms polyelectrolyte complexes with anionic polymers such as poly-
phosphates. It is well known that the electrostatic interaction between chitosan and

Figure 2 Environmental scanning electron micrographs of the surface of (a) silicate-coated chitosan beads
30 and (b) non-coated chitosan beads
polyphosphates results in gel formation. In this work, enzyme immobilisation was
achieved by dropping a chitosan solution containing catalase into a tripolyphosphate sol-
ution. The enzyme was entrapped by inclusion in the interior of the beads. Entrapment
efficiency (percent of catalase entrapped) described in equation (1) was 61 ^ 4.9% and
could be improved by optimising gel-forming conditions. Specific activity ratio of
entrapped catalase to free catalase was 7.9 ^ 1.5%. This value is not too low, consider-
ing that bovine liver catalase entrapped in chemically cross-linked chitosan beads exhib-

D.S. Yoon et al.

ited 0.18% activity of free enzyme (Çetinus and Öztop, 2003).
Continuous H2O2 removal was carried out using the catalase-entrapped chitosan
beads as described in Materials and methods. The effect of pH, temperature and
concentration of H2O2 solution on the removal efficiency was investigated. As shown in
Figure 1, a H2O2 solution was successfully treated in . 98% efficiency under various
conditions using the immobilised catalase for 24 h. However, the removal efficiency
was dependent on pH, temperature and H2O2 concentration: the H2O2 removal effi-
ciency was low at pH 4, 70 8C and 50 mM H2O2 solution. The catalase supplier indi-
cates that Terminox Ultra performs best at pH values from 6 to 10 and at a process
temperature up to 50 8C, and the enzyme performance is reduced at concentrations
higher than 30 mM.

Continuous H2O2 removal using silicate-coated beads

Decrease in the removal efficiency at low pH, high temperature and high H2O2 concen-
tration is ascribed to loss of the catalase activity under these harsh conditions. It is
known that an immobilised catalase shows catalytic activity in a broader pH and tempera-
ture range than free enzyme (Tarhan, 1995; Çetinus and Öztop, 2000). If a catalase is
further immobilised, it will probably retain activity under more harsh conditions. There-
fore, we tried to coat catalase-entrapped chitosan beads with silicate. Silicate has been
successfully used for enzyme entrapment by the sol-gel process. Recently, catalases were
successfully encapsulated in silica sol-gel glass (Vera-Avila et al., 2004). Sol-gel cer-
amics have two major drawbacks: their brittleness and narrow mesopore network. In
order to overcome these limitations, a novel entrapment method of an alginate-silicate
sol-gel matrix was developed. For example, when lipase-entrapped Ca-alginate beads
were coated with silicate, they showed a higher reusability, illustrating the effectiveness
of the coating method (Won et al., 2005).
Silicate coating of catalase-entrapped chitosan beads was performed with TMOS
as described in Materials and methods. ESEM images and EDS analysis implied that
catalase-entrapped chitosan beads were successfully coated with silicate. The surface
of the coated beads in Figure 2(a) was quite different from that of the non-coated
beads in Figure 2(b). Silicate coating reduced the specific activity ratio of catalase
entrapped to free catalase from 7.9 ^ 1.5% to 3.7 ^ 0.3%. Using the non-coated
beads and silicate-coated beads, continuous removal of H2O2 was carried out again.
The H2O2 removal efficiencies of the non-coated beads in Figure 3 were much lower
compared to those in Figure 1, because more severe conditions were applied (pH 2.0,
90 8C and 100 mM H2O2). However, when the silicate-coated chitosan beads were
used, the removal efficiency was significantly enhanced in all cases. This might be
because additional silicate coating helped the catalase to retain catalytic activity
under these severe conditions. We are expecting that optimisation of the coating
conditions will offer us even better catalase performance, resulting in higher H2O2
removal efficiency. 31
 (a) 100

H2O2 removal efficiency 98

96
D.S. Yoon et al.

94

92
pH 2.0 without coating
pH 2.0 with coating
90
0 5 10 15 20 25 30
Time (h)
(b) 100
H2O2 removal efficiency

98

96

94

92 Temp. 90oC without coating

Temp. 90oC with coating

90
0 5 10 15 20 25 30
Time (h)

(c) 100
H2O2 removal efficiency

80

60

40
100mM H2O2 without coating
100mM H2O2 with coating

20
0 5 10 15 20 25 30
Time (h)

Figure 3 Effect of silicate coating on continuous removal of H2O2 using the catalase-entrapped chitosan
beads. (a) 10 mM H2O2 solution at pH 2.0 and 20 8C; (b) 10 mM H2O2 solution at pH 7.2 and 90 8C; (c)
100 mM H2O2 solution at pH 7.2 and 20 8C

Conclusions
A commercial catalase was immobilised in chitosan beads for continuous removal of
hydrogen peroxide. The entrapment efficiency (percent of catalase entrapped) was
32 61 ^ 4.9% and the specific activity ratio of entrapped catalase to free catalase was
7.9 ^ 1.5%. Various H2O2 solutions in pH, temperature, and concentration were continu-
ously treated in a column packed with the immobilised catalase in high removal effi-
ciency for 24 h. Silicate coating of the catalase-entrapped chitosan beads greatly
improved the performance of the catalase under harsh conditions such as pH 2.0, 90 8C,
and 100 mM hydrogen peroxide.

Acknowledgements

D.S. Yoon et al.

This research was supported by the Ministry of Environment of the Republic of Korea.

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