Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation docx

Enhanced production of pectinase by Bacillus sp. DT7
using solid state fermentation
Des Raj Kashyap
a
, Sanjeev Kumar Soni
a
, Rupinder Tewari
b,
*
a
Department of Microbiology, Panjab University, Chandigarh 160014, India
b
Department of Biotechnology, Panjab University, Chandigarh 160014, India
Received 22 May 2002; received in revised form 10 August 2002; accepted 14 August 2002
Abstract
Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of
this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and
vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with
polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 ll/g dry substrate) with distilled water at 75%
moisture level, after 36 h of incubation at 37 °C.
Ó 2002 Published by Elsevier Science Ltd.
Keywords: Solid state fermentation; Bacillus; Pectinase
1. Introduction
In recent years there has been a renewed interest in
solid-state fermentation (SSF) processes for the pro-
duction of bioactive compounds. While efforts continue
largely to exploit filamentous fungi and yeasts for the
production of various enzymes, attempts have also been
made to explore possibilities of using bacterial strains in
SSF systems (Pandey et al., 2000). Enzyme production
by SSF using bacterial spp. has been reported for many

enzymes such as xylanase (Gessesse and Mamo, 1999)
and amylase (Babu and Satyanarayana, 1995) but re-
ports on pectinase production by SSF using bacterial
spp. are lacking in the literature. The use of SSF for the
production of enzymes mainly from fungi may be due to
the general belief that SSF technique is applicable only
to filamentous fungi (Lonesane and Ghidyal, 1992).
Studies on comparative production of pectinase by As-
pergillus sp. using submerged fermentation (SmF) and
SSF has shown the latter to be a better option (Solis-
Pereyra et al., 1993; Minjares-Carranco et al., 1997).
Moreover, the use of SSF has been reported to be more
advantageous than SmF as it allows cheaper production
of enzyme having better physiochemical properties than
that produced by SmF (Solis-Pereyra et al., 1993).
In this study, we report the production of very high
levels of an alkali- and thermotolerant pectinase pro-
duced by a mesophilic Bacillus sp. DT7 (Kashyap et al.,
2000) using SSF conditions.
2. Methods
2.1. Solid state fermentation
Solid substrates (5.0 g; wheat bran, rice bran, apple
pomace) in 250 ml Erlenmeyer flasks were moistened
with specified content of distilled water and autoclaved at
15 psi for 30 min. 2.0 ml of culture was used as an in-
oculum in each flask of solid substrate, which were then
incubated at 37 °C for specified time intervals. Visual
observations regarding growth were made on each day
and samples were drawn periodically to assess the en-
zyme production. Enzyme was extracted with 25 ml,

Tris–HCl buffer (0.01 M, pH 8.0) and filtered. The pooled
filtrate of two extractions was centrifuged (10,000g for 15
min; 4 °C) and used as the source of enzyme.
Pectinase activity in the extracted liquid was assayed
by the calorimetric method described earlier (Kashyap
et al., 2000). Pectinase activity in the supernatant was
expressed as U/g of solid substrate used.
*
Corresponding author. Address: Department of Microbiology,
Panjab University, Chandigarh 160014, India. Fax: +91-172-541409.
E-mail address: (R. Tewari).
0960-8524/03/$ - see front matter Ó 2002 Published by Elsevier Science Ltd.
doi:10.1016/S0960-8524(02)00206-7
Bioresource Technology 88 (2003) 251–254
2.2. Optimization studies for pectinase production
The effect of environmental parameters in SSF was
studied using wheat bran, unless otherwise stated, by
altering physiochemical and culture conditions.
Effect of different solid substrates: The effect of dif-
ferent solid substrates on pectinase production was
studied by using a variety of solid substrates (wheat
bran, rice bran, apple pomace). The original moisture
content of the apple pomace was 80%. Effect of moisture
contents on pectinase production using wheat bran and
rice bran was studied by adding different amount (50%,
60%, 66.7%, 75%, 77.8%, and 80%) of distilled water as
the moistening agent to these solid substrates.
Effect of salts: Different salts: CaCl
2
Á 2H

2
O,
MgSO
4
Á 7H
2
O, CoCl
2
Á 2H
2
O, MnSO
4
Á 4H
2
O, H
3
BO
3
,
ZnCl
2
, KCl and NaCl (1 mM, final concentration) were
dissolved in the distilled water used to adjust moisture
level in the solid substrate.
Effect of carbon sources: Different carbon sources:
glucose, mannitol, pectin, polygalacturonic acid (PGA),
galactose, sucrose, lactose, maltose, sodium acetate (SA)
and xylose were supplemented separately to a final
concentration of 1% (w/v) in solid media. They were
dissolved to required concentrations in 15 ml of distilled

water, which was used as moistening agent per 5 g of
solid substrate.
Effect of nitrogen sources: Different organic and in-
organic nitrogen sources: yeast extract, peptone, tryp-
tone, glycine, urea, ammonium chloride, ammonium
nitrate, ammonium sulphate and ammonium citrate
were supplemented separately to a final concentration of
1% (w/v) in solid media after dissolving them in distilled
water used for adjusting the moisture content.
Effect of vitamin supplementation: Effect of neurobion
(Nb) (a multivitamin solution of vitamin B
1
,B
6
and B
12
;
E-Merck, India Ltd.) on pectinase production was
studied by supplementing different concentrations (9–54
ll/g dry substrate) of Nb into the solid medium. For this
5.0 g wheat bran and 15 ml of distilled water were au-
toclaved (15 psi, 30 min) separately. Nb was then added
to the distilled water, which was subsequently used as
moistening agent.
Effect of optimized components: Effect of optimized
components on pectinase production in optimized me-
dium (wheat bran) was studied by supplementing these
components alone or in combination in distilled water
used as moistening agent.
3. Results and discussion

Research on the selection of suitable substrates for
pectinase production has mainly centered on tropical
agro-industrial crops and residues. Of the various sub-
strates reported in the literature, wheat bran has been
the prime among all (Pandey et al., 2000). In the present
study, out of three substrates (wheat bran, rice bran and
apple pomace) used wheat bran yielded 4600 U of pec-
tinase/g dry substrate at 75% moisture level after 36 h of
incubation at 37 °C. When rice bran and apple pomace
were used separately as prime substrates, maximum
pectinase yields obtained were 3265.25 and 78.0 U/g dry
substrate at 66.7 and 80% moisture contents respectively
after 48 h of incubation at 37 °C. A decline in pectinase
activity in wheat bran (26%) and rice bran (8.2%) was
observed after 72 h of incubation. Moreover, the pro-
duction of this enzyme using SSF process was much
higher than the SmF process reported previously from
the same bacteria (Kashyap et al., 2000). Higher pro-
duction of pectinase in SSF process may be due to the
reason that solid substrate not only supplies the nutrient
to the microbial cultures growing in it, but also serves as
anchorage for the cells (Pandey et al., 2000) allowing
them to utilize the substrate effectively. However, some
of the nutrients in the solid substrate may be available in
suboptimal concentrations, or even not present in the
substrates. In such cases, it would be necessary to sup-
plement them externally. Therefore, wheat bran was
supplemented with different salts (final concentration, 1
mM), carbon (final concentration, 1% w/v) and nitrogen
(1%, w/v) sources and Nb (27 ll/g dry substrate) to

evaluate their effect on pectinase production at 75%
moisture level.
Addition of CaCl
2
Á 2H
2
O and MgSO
4
Á 7H
2
O en-
hanced pectinase production by 28.0% and 11.6% re-
spectively, whereas CoCl
2
Á 2H
2
O and MnSO
4
Á 4H
2
O
did not have any effect on pectinase production (Table
1). Salts such as H
3
BO
3
, ZnCl
2
, KCl and NaCl inhibited
the pectinase production up to 12%. Carbon sources

such as PGA, SA, pectin and lactose were found to
enhance pectinase production from 11% to 44% (Table
2).
When various nitrogen sources were supplemented in
wheat bran medium, yeast extract (YE), peptone and
ammonium chloride were found to enhance pectinase
production up to 24% (Table 3). Addition of glycine,
Table 1
Production of pectinase from Bacillus sp. DT7 in wheat bran using
various salts
Salt (1 mM) Relative activity (%)
Control 100.0
CaCl
2
Á 2H
2
O 128.0
MgSO
4
Á 2H
2
O 11.5
CoCl
2
Á 2H
2
O 102.0
MnSO
4
Á 4H

2
O 104.8
H
3
BO
3
89.0
ZnCl
2
87.5
KCl 93.4
NaCl 93.0
Note: Incubated at 37 °C for 36 h incubation. 100% activity is equiv-
alent to 4600 U/g dry substrate.
252 D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254
urea and ammonium nitrate inhibited pectinase pro-
duction, which may be due to poor growth of Bacillus in
the medium containing these nitrogen sources whereas,
tryptone had no effect on pectinase production in solid
medium.
Effect of Nb on pectinase production in solid medium
was studied by supplementing different concentrations
(9–54 ll/g dry substrate) of this multivitamin solution in
wheat bran. Pectinase production was enhanced by
65.8% when 27 ll/g dry substrate of multivitamin solu-
tion was added to wheat bran medium. Increasing the
concentration of Nb above 27 ll/g dry substrate had no
effect on pectinase production.
The effect of optimized components alone or in
combinations on pectinase production by Bacillus sp.

DT7, was studied in wheat bran medium at 37 °C for 36
h. Of the different components supplemented combina-
tion of Nb (27 ll/g dry weight) and PGA (3%, w/w),
enhanced pectinase production by 75% (Table 4),
whereas combination of Nb with other components such
as YE (3%, w/w) þ PGA; PGA þ SA (3%, w/w),
YE þ PGA þ SA and PGA þ SA þ YE þ CaCl
2
Á 2H
2
O
increased pectinase production from 43% to 70% de-
pending on the combinations used.
From the present study, it is apparent that SSF
process for pectinase production from a bacterial source
is a better option than SmF. Moreover, as wheat bran is
a cheap and readily available byproduct, the production
of pectinase using SSF may be a cost-effective affair. As
this enzyme has been successfully used for degumming
of fibre crops in our previous study (Kashyap et al.,
2001), the low cost of its production may further
broaden the scopes for its use in industries involved in
treatment of fibre crops.
References
Babu, K.R., Satyanarayana, T., 1995. a-amylase production by the
thermophilic Bacillus coagulans in solid state fermentation. Process
Biochem. 30, 305–309.
Gessesse, A., Mamo, G., 1999. High level xylanase production by an
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Table 4
Production of pectinase from Bacillus sp. DT7 in wheat bran supple-
mented with optimized components
Component Relative activity (%)
Control 100
PGA 144.0
SA 140.0
YE 125.0
Nb 166.0
CaCl
2
Á 2H
2
O 129.0
PGA þ YE 143.5
PGA þ Nb 175.0
PGA þ SA 148.4
PGA þ CaCl
2
Á 2H
2
O 146.5
YE þ SA 148.4
SA þ Nb 163.0

SA þ CaCl
2
Á 2H
2
O 144.0
YE þ Nb 170.5
YE þ CaCl
2
Á 2H
2
O 130.8
PGA þ SA þ YE 149.0
PGA þ SA þ Nb 169.8
PGA þ CaCl
2
Á 2H
2
O 149.0
SA þ YE þ Nb 169.8
PGA þ YE þ CaCl
2
Á 2H
2
O 145.2
SA þ YE þ Nb 164.6
SA þ YE þ CaCl
2
Á 2H
2
O 163.0

YE þ Nb þ CaCl
2
Á 2H
2
O 167.5
PGA þ SA þ YE þ Nb 169.0
PGA þ SA þ YE þ Nb þ CaCl
2
Á 2H
2
O 170.0
Note: Incubated at 37 °C for 36 h. 100% pectinase activity is equivalent
to 4600 U/g dry substrate.
Table 2
Production of pectinase from Bacillus sp. DT7 in wheat bran using
various carbon sources
Carbon source
(final concentration, 1% w/v)
Relative activity (%)
Control 100
Pectin 117.8
Glucose 77.2
Galactose 75.5
Mannitol 104.0
Sucrose 82.4
SA 141.1
Lactose 111.3
PGA 143.8
Maltose 39.0
Xylose 64.5

Note: Incubated at 37 °C for 36 h. 100% activity is equivalent to 4600
U/g dry substrate.
Table 3
Production of pectinase from Bacillus sp. DT7 in wheat bran using
various nitrogen sources
Nitrogen source
(1%, w/v, final concentration)
Relative activity (%)
Control 100
YE 124.8
Peptone 121.4
Tryptone 102.0
Glycine 0
Urea 0
Ammonium chloride 110.7
Ammonium nitrate 0
Ammonium sulphate 76.42
Note: Incubated at 37 °C for 36 h. 100% pectinase activity is equivalent
to 4600 U/g dry substrate.
D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254 253
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254 D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254