Journal of Applied Microbiology 1997, 83, 402406

Production of alkaline xylanase by an alkaliphilic Bacillus sp.

isolated from an alkaline soda lake
A. Gessesse* and B.A. Gashe1
Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia and 1Department of Biological Sciences,
University of Botswana, Gaborone, Botswana
5956/10/96: received 21 October 1996, revised 29 January 1997 and accepted 30 January 1997

An alkaline xylanase-producing alkaliphilic Bacillus

sp. AR-009 was isolated from an alkaline soda lake in Ethiopia. The enzyme was
optimally active at pH 9 and was stable over a broad pH range. The optimum
temperature for xylanase activity, assayed at pH 9, was 6065C. Measured at
pH 8 and 9, the enzyme had good stability at 55 and 60C. At both pH values, over
80% of its original activity was retained after heating for 25 h at 55C. At 60C,
the enzyme maintained 63% of its original activity after 25 h incubation while at pH
9 it retained 54% of its original activity after 1 h heating. These properties qualify the enzyme
to be novel and potentially important for application in some industrial processes.

A . G ES S ES SE A ND B. A . G AS H E. 1997.

INTRODUCTION

Xylan, a b-1,4 linked polymer of D-xylose, is the major

constituent of plant hemicelluloses. A wide variety of microorganisms are known to produce xylanases, enzymes that are
involved in the hydrolysis of xylan (Wong et al. 1988 ; Bastawde 1992). In recent years increasing attention has been
given to the study of xylan-degrading enzymes because of
their potential application in different industrial processes.
One area of application is in the pulp and paper industry
where xylanase can be used for the bleaching of kraft pulps
(Senior et al. 1992; Viikari et al. 1994). The use of xylanase
prior to the normal bleaching operation has been shown
to significantly reduce the amount of chlorinated organic
compounds formed during the bleaching process (Senior et
al. 1992), thus reducing the risk of environmental pollution.
Since the kraft process of pulp and paper making is carried
out at alkaline pH and high temperature, the use of alkaline
xylanases with higher temperature optima is considered to be
advantageous (Zamost et al. 1991; Yang et al. 1995). Alkaline
xylanases will also find a number of other applications. For
example, because of the high solubility of xylan at alkaline
pH (Grant and Horikoshi 1992) alkaline xylanases may have
good potential for the hydrolysis of hemicellulosic wastes to
fermentable sugars.
Until now there have been very few reports on the production of alkaline xylanses (Nakamura et al. 1993a, b; Yang
Correspondence to : Dr A. Gessesse, Department of Biology, Addis Ababa
University, PO Box 1176, Addis Ababa, Ethiopia.

et al. 1995). Many xylanase-producing alkaliphilic microbial

strains have been reported from different laboratories.
However, the xylanases from most of these alkaliphilic strains
have their optimum pH around neutrality (Okazaki et al.
1984; Tsujibo et al. 1990; Dey et al. 1992; Ratto et al. 1992).
The majority of alkaliphiles known so far were isolated from
neutral soil samples. On the other hand, naturally occurring
alkaline habitats are found scattered in different parts of the
world (Grant and Horikoshi 1992). Such habitats are expected
to harbour novel micro-organisms that are adapted to living
at alkaline pH. Extracellular enzymes produced by such
organisms are likely to have their optimum pH for activity
in the alkaline range. Such enzymes may find important
application in different industrial processes. Until now there
has been very little effort to isolate alkaliphiles from naturally
occurring alkaline habitats. In the present study, the production of an alkaline xylanase by an alkaliphilic Bacillus sp.
isolated from an alkaline soda lake and the properties of the
crude enzyme are reported.
MATERIALS AND METHODS
Isolation and screening

Water samples were collected from lake Arenguadie, an alkaline soda lake found near the town of Debre Zeit, central
Ethiopia. A loop full of the sample was streaked onto xylancontaining nutrient agar plates. The pH of the medium was
adjusted to 103 by adding 1% sodium carbonate. After 48 h
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P RO DU C TI ON O F A LK A LI NE X YL AN A SE 403

35
30

25
3

20

15
10

1
5

Enzyme production

The growth medium used for xylanase production was composed of (g l1): xylan, 5; peptone, 5; yeast extract, 1;
K2HPO4, 1; MgSO4 . 7H2O, 02; CaCl2, 01; and Na2CO3, 10.
Sodium carbonate was autoclaved separately and added to
the rest of the medium after cooling. The above medium (100
ml in 500 ml baffled flasks) was inoculated with 2 ml of an
overnight culture and incubated at 35C in a rotary incubator
shaker. The culture was harvested at the stationary growth
phase and centrifuged at 5000 g. The cell-free supernatant
fluid was used as the enzyme source.
Partial purification of the enzyme

The cell-free supernatant fluid was precipitated with the

addition of solid ammonium sulphate to 70% saturation.
After centrifugation the pellet was suspended in a minimum
volume of 50 mmol l1 glycine-NaOH buffer, pH 9, and
dialysed against three changes of the same buffer. The dialysed crude enzyme preparation was used for all subsequent
studies.
Enzyme assay

Xylanase activity was assayed by measuring the release of

reducing sugar from birch wood xylan following the dinitrosalicylic acid (DNS) method (Miller 1959). To 09 ml of
substrate in 50 mmol l1 glycine-NaOH buffer, pH 9, 100 ml
of appropriately diluted enzyme was added and incubated at
50C. After 10 min, 15 ml of DNS solution was added to
the reaction mixture and boiled for 5 min. Absorbance was
measured at 540 nm against a reagent blank. One unit of
xylanase activity was defined as the amount of enzyme that
released 1 mmol reducing sugar equivalent to xylose per min
under the above assay conditions.
RESULTS
The organism

AR-009 was isolated from an alkaline soda lake water sample

collected from lake Arenguadie. It grows at pH 10105 and

0
0

10

20

30
Time (h)

40

50

Enzyme activity (U ml1)

Biomass (O.D. 600)

incubation at 35C, individual colonies were transferred to

fresh xylan-containing nutrient agar plates and incubated as
above. Xylanase-producing strains were selected by flooding
the plates with 01% aqueous Congo red for 15 min followed
by repeated washing with 1 mol l1 NaCl. All colonies showing a clear zone on agar plates were further screened by
growing them in liquid medium and assaying enzyme activity
from the cell-free culture supernatant fluid. One strain, designated as AR-009, was selected for subsequent studies.

0
60

Fig. 1 Time course of growth and enzyme production by Bacillus

sp. AR-009. Cells were grown in a medium containing birch wood

xylan, pH 103. Growth was monitored by measuring optical
density at 600 nm (O.D. 600). Xylanase activity was assayed
from the cell-free culture supernatant fluid at 50C, pH 9, ,
O.D. 600; , enzyme activity (U ml1)

produced a high level of xylanase activity both in solid and

liquid media. The organism was rod-shaped, Gram-positive,
aerobic, motile, an endospore-former, with terminally located
spores and catalase-positive. On the basis of these properties
it was classified as a strain of the genus Bacillus (Claus and
Berkeley 1986).
Enzyme production

Xylanase production by AR-009 was growth-associated,

reaching a maximum after 20 h (Fig. 1). Enzyme production
remained more or less the same up to 56 h while biomass
started to gradually decline after 40 h. A high level of enzyme
production was observed when the organism was grown in
media containing oat spelt xylan, wheat bran and birchwood
xylan as carbon sources (Table 1). A significant amount of
Table 1 Effect of different carbon sources on xylanase

production by Bacillus sp. AR-009

Sugar
Xylanase activity (U ml1)

Oat spelt xylan

452
Birchwood xylan
350
Wheat bran
446
Starch
210
Sucrose
146
Arabinose
141
Glucose
106
Xylose
60

Cells were grown for 40 h in the presence of 05% carbon source.

Xylanase activity was measured from the cell-free supernatant fluid
at pH 9 and 50C.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

404 A . G ES S ES SE A ND B. A . G AS H E

The effect of pH on xylanase activity was measured at 50C

by varying the pH of the assay medium. Over 90% of the
maximum activity was in the pH range of 7595 with an
optimum at pH 9 (Fig. 2). At pH 10, 67% of the maximum
activity was retained. To study the effect of pH on stability,
the crude enzyme was diluted in different buffers of varying
pH values and incubated at 50C for 1 h. Residual activity
was measured following the standard assay method. The
enzyme was very stable in the pH range of 610 (Fig. 3).
The temperature profile of AR-009 xylanase was studied
by assaying enzyme activity at different temperatures using
pH 9 buffer. The optimum temperature was 6065C (Fig.
4). The effect of temperature on enzyme stability was determined at 55 and 60C by incubating the enzyme in pH 8
and 9 buffer. At 55C, the enzyme was very stable at both
pH values maintaining over 80% of its original activity after
25 h heating (Fig. 5). At 60C, 54% of its original activity
was retained after 1 h incubation at pH 9 while 63% of the
original activity was maintained after 25 h heating at pH 8
(Fig. 5).
DISCUSSION

Bacillus sp. AR-009 produced a high level of xylanase in the

presence of xylan and wheat bran as carbon sources. An

Residual activity (%)

Properties of the crude enzyme

100
80
60
40
20
0
5

8
pH

10

11

Fig. 3 Effect of pH on xylanase stability. The enzyme was

diluted with different buffers of varying pH values and incubated

at 50C for 1 h. Residual activity was assayed at pH 9 and 50C.
Buffers used were: , citrate; , phosphate and t, glycine-NaOH

100
Relative activity (%)

xylanase was also produced when starch, sucrose, arabinose,

glucose and xylose were used as carbon sources (Table 1).
However, compared with xylan and wheat bran, these sugars
induced lower xylanase activity.

80
60
40
20
0
30

40

50
60
Temperature (C)

70

Fig. 4 Effect of temperature on xylanase activity. The reaction

was carried out at pH 9 for 10 min

Relative activity (%)

100
80
60
40
20
0
6

10

12

pH

Fig. 2 pH profile of Bacillus sp. AR-009 xylanase. Enzyme

activity was measured at 50C and different pH values. The

buffers used, each at a concentration of 50 mmol l1, were : ,
citrate; , phosphate; t, Tris-HCl and T, glycine-NaOH

appreciable level of xylanase activity was also produced in the

presence of other carbon sources including xylose. This may
indicate that isolate AR-009 produced multiple forms of xylanase whereby one form is produced constitutively in small
amounts and the other is induced by the substrate xylan.
Many xylanase-producing microbial strains have been shown
to produce multiple forms of xylanase (Honda et al. 1985;
Wong et al. 1988; Elegir et al. 1994; Flint et al. 1994).
Xylanases that are thermostable and have their optimum
pH in the alkaline range are considered to have good potential
for application in the pulp and paper industry. This is because
the use of such enzymes is expected to greatly reduce the
need for costly pH and temperature readjustments before

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

P RO DU C TI ON O F A LK A LI NE X YL AN A SE 405

REFERENCES

Residual activity (%)

100
80
60
40
20
0
0

50

100
Time (min)

150

Fig. 5 Thermal stability of Bacillus sp. AR-009 xylanase. The

crude enzyme preparation was diluted in 50 mmol l1 TrisHCl buffer, pH 8, (, t), or 50 mmol l1 glycine-NaOH
buffer, pH 9 (, T), and incubated at 55C (, ) and
60C (t, T). Samples were withdrawn at time intervals and
residual activity measured at 50C, pH 9

enzyme addition. Most xylanases known so far have their

optimum pH around neutrality. Even xylanases produced by
most alkaliphiles reported to date have their optimum pH
around neutrality (Okazaki et al. 1984; Rajarama and Varma
1990; Tsujibo et al. 1990; Dey et al. 1992; Park et al. 1992).
Nakamura et al. (1993a, b) reported the first alkaline xylanase
produced by Bacillus sp. strain 41M-1 which had an optimum
temperature and pH of 50C and 9, respectively. The thermal
stability of this enzyme was low, losing about 90% of its
activity after 30 min heating at 60C, pH 9. Yang et al. (1995)
isolated an alkaliphilic Bacillus sp. V1-4 from a hard wood
kraft pulp which produced a xylanase having a pH optimum
of 685 and a temperature optimum of 55C. At 60C and
pH 9, this enzyme was reported to retain 1520% of its
original activity after 30 min incubation. The xylanase from
Bacillus sp. AR-009 was a novel enzyme, being active at
alkaline pH with an optimum at pH 9 and was stable over a
broad pH range. It showed optimum activity at 6065C
and good stability at 55 and 60C at alkaline pH values.
These are desirable properties for application in the pulp and
paper industry as well as in other industrial processes.

ACKNOWLEDGEMENTS

This work was supported by a grant from Sida-SAREC

(Sweden) administered through the Ethiopian Science and
Technology Commission. The authors thank Ato Gashaw
Mamo for valuable discussions.

Bastawde, K.B. (1992) Xylan structure, microbial xylanases, and

their mode of action. World Journal of Microbiology and Biotechnology 8, 353368.
Claus, D. and Berkeley, R.C.W. (1986) Genus Bacillus. In Bergeys
Manual of Systematic Bacteriology Vol. 2. ed. Sneath, P.H.A. pp.
11051138. London : Williams and Wilkins.
Dey, D., Hinge, J., Shendye, A. and Rao, M. (1992) Purification
and properties of extracellular endoxylanases from alkalophilic
thermophilic Bacillus sp. Canadian Journal of Microbiology 38,
436442.
Elegir, G., Szakacs, G. and Jefferies, T.W. (1994) Purification,
characterization, and substrate specificities of multiple xylanases
from Streptomyces sp. strain B-12-2. Applied and Environmental
Microbiology 60, 26092615.
Flint, H.J., Zhang, J.-X. and Martin, J. (1994) Multiplicity and
expression of xylanases in the rumen cellulolytic bacterium Ruminococcus flavefaciens. Current Microbiology 29, 139143.
Grant, W.D. and Horikoshi, K. (1992) Alkaliphiles : ecology and
biotechnological applications. In Molecular Biology and Biotechnology of Extremophiles ed. Herbert, R.A. and Sharp, R.J. pp.
143162. New York : Chapman and Hall.
Honda, H., Kudo, T., Ikura, Y. and Horikoshi, K. (1985) Two
types of xylanases of alkalophilic Bacillus sp. No. C-125. Canadian
Journal of Microbiology 31, 538542.
Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry 31, 426428.
Nakamura, S., Wakabayashi, K., Nakai, R., Aono, R. and Horikoshi,
K. (1993a) Purification and some properties of an alkaline xylanase
from alkaliphilic Bacillus sp Strain 41M-1. Applied and Environmental Microbiology 59, 23112316.
Nakamura, S., Wakabayashi, K., Nakai, R. and Horikoshi, K.
(1993b) Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1. World Journal of Microbiology
and Biotechnology 3, 221224.
Okazaki, W., Akiba, T., Horikoshi, K. and Akahoshi, R. (1984)
Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus sp. Applied Microbiology and Biotechnology 19, 335340.
Park, Y.S., Yum, D.Y., Bai, D.H. and Yu, J.H. (1992) Xylanase
from alkaliphilic Bacillus sp. YC-335. Bioscience Biotechnology
Biochemistry 56, 13551356.
Rajarama, S. and Varma, A. (1990) Production and characterization
of xylanase from Bacillus thermoalkalophilus grown on agricultural
wastes. Applied Microbiology and Biotechnology 34, 141144.
Ratto, M., Poutman, K. and Viikari, L. (1992) Production of xylanolytic enzymes by an alkalitolerant Bacillus cirulans strain.
Applied Microbiology and Biotechnology 37, 470473.
Senior, D.J., Hamilton, J., Bernier, R.L. and Manoir, J. (1992)
Reduction in chlorine use during bleaching of kraft pulp following
xylanase treatment. Tappi Journal 75, 125130.
Tsujibo, H., Sakamoto, T., Nishino, N., Hasegawa, T. and Inamori,
Y. (1990) Purification and properties of three types of xylanases
produced by an alkaliphilic actinomycete. Journal of Applied Bacteriology 69, 398405.
Viikari, L., Kantelinen, A., Sundquist, J. and Linko, M. (1994)
Xylanases in bleaching : from an idea to the industry. FEMS
Microbiology Review 13, 335350.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406

406 A . G ES S ES SE A ND B. A . G AS H E

Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N. (1988) Multiplicity

of b-1,4-xylanase in microorganisms : functions and applications.
Microbiological Reviews 52, 305317.
Yang, V.W., Zhuang, Z., Elegir, G. and Jeffries, T.W. (1995)
Alkaline-active xylanase produced by an alkaliphilic Bacillus

sp isolated from kraft pulp. Journal of Industrial Microbiology 15,

434441.
Zamost, B.L., Nielsen, H.K. and Starnes, R.L. (1991) Thermostable
enzymes for industrial applications. Journal of Industrial Microbiology 8, 7182.

1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 83, 402406