Production of Bacterial Pectinase(s) from Agro-Industrial Wastes Under Solid State

Journal of Applied Sciences Research, 4(12): 1708-1721, 2008
© 2008, INSInet Publication
Production of Bacterial Pectinase(s) from Agro-Industrial Wastes Under Solid State
Fermentation Conditions
1
1
Reda, A. Bayoumi, 2Hesham, M.Yassin, 2Mahmoud, A. Swelim, 2Ebtsam,Z.Abdel-All
Botany & Microbiology Department, Faculty of Science, Al-Azhar University,Cairo,Egypt.P.O.11884.
2
Botany Department, Faculty of Science , Benha University, Egypt.
Abstract: Bacillus firmus –I-4071 produced very high level of polygalacturonase by solid state
fermentation (SSF). Fifty one bacterial isolates were isolated from fermented clayed Solanum tuberosum
peels collected from different restaurants in Kalubeia governorate, Egypt. All bacterial isolates were
screened for their ability to produce pectinases using apple pectin under solid state fermentation (SSF)
conditions. The results showed that all of these isolates were found to have appreciable pectinolytic
productivities of which twenty isolates showed good pectinases-producing potentialities using agroindustrial wastes viz. Solanum tuberosum (ST), Solanum melanogena (SM), Echornia crasips (EC) and
citrus peels mixture (CPM) at 30 °C and pH 6 by pectin clearing zone (PCZ) technique. Three bacterial
isolates, viz: 4071, 107 and 10104 were found to exhibit a higher polygalacturonase (PG) production by
attacking Solanum tuberosum (ST) peels compared to other wastes. The three most potent bacterial isolates
were identified on the bases of cell shape, cell arrangement, relation to oxygen and physiological and
biochemical tests as Bacillus firmus, I-4071, B. firmus-I-10104 and Bacillus laterosporus-I-107. The
optimum inoculum size for production of polygalacturonase production by B. firmus-I-10104 on Solanum
tuberosum (ST ) peels was 1 ml (30 x 10 1 5 CFU); substrate concentration, 1.25 g/25 ml; incubation period,
96 hours, pH, 6.0; incubation temperature, 37 °C; different nitrogen source; peptone (0.1 g/l); different
carbon source, control; different amine acids, control and finally without any vitamins. These results
suggesting that, the polygalacturonase was produced from cheap raw material under solid state
fermentation under all optimal conditions for application in the clarification of juice.
Key words: Polygalacturonase(PG)production,
Agro-Industrial wastes
Bacillus
INTRODUCTION
Most of the chemical changes that occur in living
tissues are regulated by enzymes. In recent years there
has been a renewed interest in solid-state fermentation
(SSF) processes for the production of bioactive
compounds. Enzymes production by SSF using
bacterial spp. has been reported for many enzymes
such as xylanase [1 7 ] and amylase [3 ] but reports on
pectinase production by SSF using bacterial spp. are
lacking in the literature. SSF has been reported to be
more advantageous than submerged (SmF) as it allows
cheaper production of enzyme having better
physiochemical properties than that produced by
SmF [2 5 ]. Pectinases comprises a heterogeneous group of
enzymes that catalyze the breakdown of pectinc o n t a i n i n g su b s trates . P ectic s u b s tanc e s a re
characterized by long chains of galacturonic acid
residues. On these residues are carboxyl groups, which
are sometimes modified by the addition of methyl
groups, forming methoxyl groups. Pectic enzymes act
firmus,
Solid
State
Fermentation (SSF),
by breaking glycosidic bonds of the long carbon chains
(polygalacturonase, pectin lyase and pectate lyase) and
by splitting off methoxyl groups (pectin esterase)
[1 0 ,1 1 ,1 2 ;1 9 ;4 ]
. Pectic substances are widely distributed in
fruits and vegetables [1 0 -3 0 % ] in turnips, peels of orange
and in pulps of tomato, pineapple and lemon), hence
they form important natural substrates for pectinases
[1 9 ]
. Enzymes which degrade pectic substances are
pectinases or pecteolytic enzymes and can be classified
into three types. Pectin methyl esterase (PME)
hydrolyzes the methyl ester of galacturonide chain
liberating methanol. Polygalacturonases (PGases) and
pectate lyases (PLases) split the molecular chains of
the respective polymers [1 8 ,1 ,2 4 ]. Since the 1940s,
pectinases have been exploited for many industrial
applications. Pectinases are mainly used for increasing
filtration efficiency and clarification of fruit juices, in
wood preservation and used in maceration, liquefaction
and extraction of vegetable tissues [9 ,4 ]. Various
literature reports and reviews are available on the
production and applications of pectinases [4 2 ,2 4 ]. Also,
Corresponding Author: Reda Ahmed Bayoumi, Bayoumi, R.A., Botany & Microbiology Department, Faculty of Science, AlAzhar University, Cairo, Egypt. P.O.11884.
Tel.: +(0020) 24108598;
Fax:+(0020) 22629356. Mobile: 0103597140
E-mail address: [email protected]
1708
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
pectinases have been used in the paper and pulp
industry in addition to cellulases [4 0 ], and plant
pathology [2 8 ]. Few reviews have highlighted the
biological and technological importance of pectinases [4 0 ,
2 4 ,1 5 ,2 ,1 4 ]
.
In this study, we report the nutritional and
environmental conditions requirements for production
of PG by Bacillus firmus-I-4071 under solid state
fermentation conditions using potato peels.
M ATERIALS AND M ETHODS
1. Isolation of Bacteria: Bacterial isolates were
obtained from clayed potato peels collected from
different restaurants in Benha, Kalubia governorate,
Egypt. The potato peels were applied using the soil
dilution plate method.
2. Construction of Galacturonic Acid Standard
Curves: A stock solution (10.000 mg/ml) of the
standard galacturonic acid supplied by Sigma was
prepare in acetate buffer (0.2 M ) at pH 5. The stock
so lutio n was used fo r making o f diffe re nt
concentrations. After preparing of the required
dilutions, only 1.0 ml of each dilution was transferred
to determine of the amount of reducing sugar according
to Nelson [3 3 ]. A standard curve was constructed
relating all different sugar concentrations applied
against their corresponding to optical density at 510
nm. The obtained standard curve was used for
estimating the polygalacturonase activities in terms of
mg/ml and then units (U). One unit is defined as the
amount of enzyme protein (mg) required to exert free
galacturonic acid, from pectin of time under 35 °C for
1hour in acetate buffer (0.2 M) at pH 5.0
.
3. Protein Determination: Protein of all enzymatic
preparations was determined by the method of Lowry
et al., [3 1 ].
4. Growth and M aintenance M edium: a-Czapek'sDox pectin medium:
This medium contained (g/l): Pectin, 10; NaNO 3 ,
2; K H 2 PO 4 , 1; KCl, 0.5; M gSO 4 .7H 2 O, 0.5;
FeSO 4 .7H 2 O, 0.001%, CaCl2 , 0.001%, agar-agar, 15;
and distilled water up to 1000 ml. pH was adjusted at
7, and then autoclaved for 20 minutes at 1.5
atmospheric pressure. This medium was modified as
follow: Part (A) contained (g/l): NaNO 3 , 2; KH 2 PO 4 ,
1; KCl, 0.5; M gSO 4 .7H 2 O, 0.5; yeast extract, 1; agaragar, 20. This contents were dissolved in 200 ml
citrate-phosphate buffer at pH 7. Part (B) contained
(g/l): Pectin, 5; dissolved in 200 ml citrate phosphate
buffer at pH 7. Part (C) citrus peels extract which
contained (g/l): Citrus peels, 125 g, the pH of the
extract was adjusted at 7 before sterilization. The three
parts (A), (B), and (C) were autoclaved for 20 minutes
at 1.5 atmospheric pressure and mixed together after
sterilization. The medium was poured into sterilized
plates, each plate contained 20 ml, the media were
allowed to be solidify.
5. Agro-industrial W astes: The potato peels wastes
were collected from different restaurants in Benha,
Kalubia governorate, Egypt. They washed to remove
the clay for using, dried in open air, and then grounded
in the production media. Citrus peels, Solanum
tuberosum (ST) peels, and water hyacinth (Echornia
crassips) were collected, dried and prepared in the
form of a ground preparations.
6. Production M edia:
6.1. Basal M edium: The basal medium (BM) was
prepared according to Vincent [5 1 ]. It contained of the
following (g/l): Sucrose, 10; KNO 3 , 0.6; KH 2 PO 4 , 1;
MgSO 4 , 0.25 and CaCl 2 , 0.1 was found most
convenient for the production of different enzymes. It
was modified to include the following constituents:
(g/l) NaNO 3 , 2; K 2 HPO 4 , 0.5; KCl, 0.5 and yeast
extract, 1. These previously mentioned contents were
dissolved in citrate phosphate buffer at pH 7.
6.2 Potato Peels Basal M edium (PPM A): It contained
the same constituents of BM supplemented with potato
peels (4 % w/v).
7. Identification of the M ost Potent Bacterial
Isolates: The three most potent bacterial isolates (B10104, B-107 and B-40710) were identified by
examination of their morphological physiological and
biochemical characteristics according to Barrow and
Feltham [5 ] ; Parker, [3 7 ]; Collee et al., [1 2 ]; Sneath
[4 9 ]
;Hensyl, [2 0 ] and Schallmey et al., [4 3 ].
8.qualitative Screening Test M edia, M ethods, and
Conditions (First Survey):
8.1. Pectinolytic Enzyme Production M edium: This
medium consists of part (A) and part (B).
Part (A) contained (g/l): NaNO 3 , 2; KH 2 PO 4 , 1; KCl,
0.5; MgSO 4 .7H 2 O, 0.5; Yeast extract, 1. These contents
were dissolved in 40 ml distilled water. The pH was
adjusted at pH 7 by NaOH (5% , w/v).Part (B)
contained (g/l): Pectin, 5, dissolved in 10 ml. of
distilled water.The two parts (A) and (B) were
autoclaved for 20 minutes at 1.5 atmospheric pressure
and mixed together after autoclaving. This medium was
inoculated with bacterial isolates. This medium was
incubated at 37 °C for 96 hours, then assayed for
pectinolytic productivity and activity were tested in the
pectinase assay medium.
8.2. Pectinase Production and Activity Assay
M edium: This medium was contained (g/l): Pectin, 1;
Arabic gum, 5; agar-agar, 15, these contents dissolved
in citrate phosphate buffer at pH 6. It was autoclaved
1709
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
at 1.5 atmospheric pressure for 20 minutes. Plates of
the same size were poured with equally amounts of
pectinase assay medium in each Petri dish. After
cooling, three wells were made by sterilized cork porer
in each plate. Each well was inoculated with 0.1 ml of
filtrate which prepared by the filtration of the broth
which grown on pectinolytic enzyme production
medium by filter paper. These plates were inoculated
at 37 °C for 24 hours. Then clearing zones of the
medium after addition of Logule's iodine solution were
investigated and taken as criteria for determining the
pectinolytic productivity
10.3. Different Incubation Periods:The most potent
bacterial isolate was allowed to grow on the waste and
incubated for 6, 12, 24, 48, 72, 96, 120, 144, 168 and
192 hours respectively.
.9.a. Qualitative Screening Test M edia, M ethods and
Conditions (Second Survey):
9.a.1. Pectinolytic Enzyme Production M edium
(Pepm): This medium contained the main ingredients
of BM supplemented with potato peels, Solanum
tuberosum (ST) peels, Echornia crassips and citrus
peels mixture (2% w/v) separately. The pH of this
medium was adjusted at 7 by dissolve its contents in
citrate-phosphate buffer (pH 7). It was autoclaved at
1.5 atmospheric pressure for 20 minutes. T his medium
was inoculated with bacterial isolates under study. This
medium was inocubated at 37 °C for 96 hours, then
assayed fo r pectin o lytic p ro d uctivity in the
polygalactunase assay medium as previously mentioned
10.5. Different Temperatures: The most potent
bacterial isolate was allowed to grow on the grounded
potato peels (PP) medium at different temperatures viz.,
10, 15, 30, 37, 40, 45, 55, 65 and 75 °C respectively
for 96h.
.9.b.medium Used in Screening Test for Selecting
the M ost Potent Bacterial Isolates: This medium
contained the main ingredients of PEPM. The
pectinolytic activity was detected by Nelson's technique
[3 3 ]
.This mixture was incubated at 45 °C for 20
minutes, assay for reducing sugar by Nelson's technique
as galacturonic acid. This mixture was modified as
follow: This mixture was incubated at 35 °C for 1 hour
.
9.c. Analysis of Reducing Sugar by Nelson's
Technique [3 3 ]:
10-parameters Controlling the Polygalacturonase
Productivities:
10.1. Different Inoculum Sizes: Different inoculum
sizes of heavy spore suspension of the most potent
bacterial isolate (prepared by harvesting 5 slants in 100
ml sterile saline solution under aseptic conditions) are
used. The following inoculum sizes were applied viz.,
1, 2, 5, 10, 15, 20, and 24 ml per each flask (250 ml).
At the end of incubation periods, polygalacturonase
productivity was determined for each flask after
incubation period as the previously mentioned.
10.2. Different Substrate Concentrations: Different
concentrations of substrate (g/flask, 25 ml, w/v) were
applied viz., 0.1, 0.3, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 5.0,
10 and 15). At the end of incubation period, enzyme
productivity were assayed.
10.4. Different pH Values:The production medium for
most potent isolate were prepared as previously
mentioned. The pH was adjusted at different pH values
viz, [7 .5 , 8 .5 , 9 .0 ] by using boric acid-borax buffer and (3,
5, 5.5, 6, 6.2, 6.4, 6.6 and 7.0) by using citratephosphate buffer. Inoculation and incubation conditions
were carried as previously mentioned.
10.6. Different Nitrogen Sources: Production medium
was supplemented with different nitrogen sources at an
equimolecular amount of nitrogen that present in
sodium nitrate (0.2%, w/v) in basal medium. Peptone,
gelatin, and casein were introduced as organic nitrogen
source at the level of 2 % and the control was devoid
from any nitrogen source. The applied nitrogen sources
were ammonium sulphate, ammonium molybdate,
ammonium chloride, ammonium oxalate, ammonium
citrate, ammonium tartrate, ammonium nitrate,
diammonium hydrogen phosphate, potassium nitrate,
gelatin, peptone, casine and urea. All other factors
(temperature, pH, substrate concentration and carbon
sources) were carried out as previously mentioned.
10.7. Different carbon sources: Different carbon
sources were introduced into the production medium at
an equimolecular amount located at 1% (w/v) glucose.
Parallel experiment was made with no sugar as a
control. The carbon sources were represented by
xylose, arabinose, glucose, galactose, mannose,
fructose, trehalose, lactose, maltose, sucrose, starch,
cellulose and pectin. Starch, cellulose and pectin was
introduced at the level of 1% (w/v). In all cases, other
previously mentioned optimal conditions were taken
into consideration.
10.8. Different Amino Acids: The production medium
was used after applying all of the previously mentioned
optimal environmental and nutritional conditions for
polygalacturonase productivity by the most potent
bacterial isolate. The used amino acids were added at
an equimolecular amount of nitrogen located in the best
inorganic nitrogen source for the enzyme productivity.
This experiment was controlled by performing of
parallel one containing the original nitrogen source i.e.,
sodium nitrate. The control of this experiment was
1710
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
devoid of any amino acid, and the supplemented amino
acids were: alanine, histadine, phenyl alanine, aspartic
acid, glycine, lysine, cystine, tryptophan, methionine,
arginine and isoleucine. Inoculation, incubation
conditions, and measurement o f the enzyme
productivity were performed as previously mentioned.
10.9. Different Vitamin Requirements: Different
vitamins viz, ascorbic acid, riboflavin, vitamin B6 and
folic acid were added separately to the medium
specialized for polygalacturonase production at 100,
250, 500, and 1000 ppm while the control was applied
free from any vitamin. Inoculation, incubation
conditions, and measurement of enzyme productivity
were performed as previously mentioned.
10.10. Different Aeration Conditions: This experiment
was carried out to investigate the effect of different
aeration conditions on polygalacturonase productivity.
It was performed by using four different volumes viz,
100, 250, 500 and 1000 ml for agricultural waste as
substrate. Each flask contained 25 ml of the medium in
case of potato peels production medium for
polygalacturonase. The enzyme productivity was
assayed as previously mentioned.
Results Fifty one bacterial isolates were isolated from
fermented Solanum tuberosum (ST) peels. These
isolates were purified, and subjected to a screening in
order to examine their pectinolytic productivities on the
basis of mean diameters of clearing zones (mm). The
fifty one bacterial isolates had a pectinolytic activity
while twenty isolates of them were considered to give
good producers. T he twenty isolates to attack some
agriculture, and industrial wastes under solid state
fermentation (SSF) The three bacterial isolates numbers
4071, 107 and 10104 out of the twenty isolates gave a
higher pectinase productivity by attacking Solanum
tuberosum (ST) peels compared to other wastes and
other isolates, where it reached up to 3.2, 3.4 and
3.4mm respectively. Bacterial isolates numbers 4071,
107 and 10104 also gave a higher polygalacturonase
productivity by attacking Solanum tuberosum (ST)
peels compared to other wastes and other isolates,
where it reached up to 515, 515 and 367.5 (U/ml)
respectively. Bacterial isolates number 4071, 107 and
10104 gave a higher polygalacturonase productivity by
attack Solanum tuberosum (ST) peels compared to
other wastes, where it reached up to 292, 287 and 297
(U/ml) respectively (Tables 1&2).
Identification of the Three M ost Potent Bacterial
Isolates: The three most potent bacterial isolates 4071,
10104 and 107 were subjected to an identification
program to the species level. The morphological
characteristics and stain reaction led to the fact that the
three bacterial isolates are suggestive to being
belonging to the genus Bacillus, gram positive aerobes
to facultative anaerobes. Consulting Bergey's Manual of
systematic bacteriology [4 9 ], the three isolates are
belonging to species firmus, firmus, and laterosporus.
They could be give the tentative name Bacillus firmus
I-4 071, Bac illus firm us-I-10104 and Bacillus
laterosporons-I-107.
Parameters Controlling the Polygalacturonase (PG)
Productivity:
1- Different Inoculum Size: Different volumes of
bacterial spore suspension were used as an inocula
sizes to inoculate flasks (250 ml) containing the
inoculum size used were 1, 2, 5, 10, 20 and 24 ml.
Each one ml of the heavy bacterial cell suspension
contained abut 30 x 10 1 5 CFU. The optimal inoculum
sizes need to produce the highest yield of
polygalacturonase were 1.0 ml. At this particular
inoculum size, the highest yield of polygalacturonase
was attained with Solanum tuberosum (ST) peels as
350 U/ml. Inoculum size above the previously optimal
recorded value gave value gradually decreasing as
compared to that of the optimum one(Figure 1).
2- Different Substrate Concentrations: The maximum
polygalacturonase productivity 437.5 U/ml was obtained
in presence of 1.25g /25 ml on Solanum tuberosum
(ST) peels by Bacillus firmus-I-10104 at 37 °C for 96h
(Figure 2).
3- Different Incubation Periods: Effect of different
inc ub atio n p erio d s o n the po lygalacturo na se
productivity using Solanum tuberosum (ST) peels under
solid state fermentation conditions by Bacillus firmus-I10104 was tested at time intervals of 6, 12, 24, 48, 72,
96, 120, 144, 168 and 192 hours. The level of
polygalacturonase increased gradually with increasing
the incubation period up to a maximum of 96h. Then
gradually decreased after these periods (Figure 3).
4- Different Initial PH Values: The polygalacturonase
productivity by Bacillus firmus-I-10104 reached its
maximum at initial pH 6.0 and 6.2. Since the enzyme
yield reached up to 325 U/ml., below and above this
optimal pH value, the enzyme productivity gradually
decreased (Figure 4).
5- D ifferent Incubatio n T em peratures: T he
polygalacturonase productivity reached its optimal value
310 U/ml at an incubation temperature at 37 °C
(Figure 5).
1711
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
Table 1: Q uantitative data of the pectinase(s) productivities of the best twenty bacterial isolates grown on four agro-industrial wastes.
Polygalacturonase productivity was detected by Nelson's technique (33) (Second Survey).
N o.
Code no. of bacterial isolates Polygalacturonase productivity (U /m l)
-------------------------------------------------------------------------------------------------------------------------------Citrus peels
Solanum tuberosum
Solam um m elongena
Echornia
m ixture (CPM )
(ST) peels
(SM ) peels
craspies (EC)
1
30107
170
335
65
0
2
3069
170
395
0
0
3
3062
170
515
35
0
4
4071
135
515
270
0
5
106
70
200
0
0
6
3034
135
362.5
0
0
7
107
130
367.5
200
0
8
3011
70
235
0
0
9
1032A
100
235
0
0
10
3084
0
235
65
0
11
4087
85
352
132
0
12
3019
135.7
80
42
0
13
30102
100
350
117.5
100
14
1032B
95
187.5
0
0
15
3050
92
342.5
120
0
16
10104
200
515
305
57.5
17
109
7.5
0
0
0
18
3056
100
305
270
127.5
19
1046
150
167.5
72.5
60
20
3048
120
325
205
25
Table 2:
N o.
1
2
3
Q uantitative data of the polygalacturonase productivity of the best three bacterial isolates grown on three agro-industrial wastes.
Polygalacturonase productivity was determ ined by N elson's technique (33).
Code no. of bacterial isolates
Polygalacturonase productivities (U /m l)
----------------------------------------------------------------------------------------------------------------------------Citrus peels m ixture (CPM )
Solanum tuberosum (ST) peels
Solanum m elanogena (SM ) peels
107
105 ± 0.05
287.5 ± 0.02
75 ± 0.01
4071
95 ± 0.09
292.5 ± 0
40 ± 0.04
10104
115 ± 0.04
297.5 ± 0
177.5 ± 0
Fig 1: Effect of different inocula sizes on the polygalacturonase (PG) productivity using Solanum tuberosum
(ST) peels under solid state fermentation (SSF) conditions by Bacillus firmus-I-10104.
1712
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
Fig 2: Effect of different substrate concentrations on the polygalacturonase productivity using Solanum
tuberosum (ST) peels under solid state fermentation conditions by Bacillus firmus-I-10104.
Fig 3: Effect of different incubation periods on the polygalacturonase productivity using Solanum tuberosum
(ST) peels under solid state fermentation conditions by Bacillus firmus-I-10104.
1713
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
Fig 4: Effect of different pH values on the polygalacturonase productivity using Solanum tuberosum (ST) peels
under solid state fermentation conditions by Bacillus firmus-I-10104.
Fig 5: Effect of different temperatures on the polygalacturonase productivity using Solanum tuberosum peels
(STP) under solid state fermentation conditions by Bacillus firmus-I-10104.
6- Different Nitrogen Sources: Effect of different
organic as well as inorganic nitrogen sources on
polygalacturonase productivity by the most potent
bacterial strain Bacillus firmus-I-10104 were studied.
Fourteen different nitrogen sources were applied as
equimolecular amount located in sodium nitrate. The
maximum value of polygalacturonase productivity
reached up to 350 U/ml in the presence of peptone
followed by ammonium chloride, urea and beef extract
(Figure 6).
7- Different C arbon Sources: The effect of thirteen
different carbon sources were introduced into the
applied production medium of polygalacturonase
productivity by Bacillus firmus-I-10104 under SSF
condition were studied. It was clear that all the
different carbon sources exhibited various degrees
lower than control by the Bacillus firmus-I-10104.
Solanum tuberosum (ST) peels was the best carbon
source for polygalacturonase production where the
productivity reached up to 115 U/ml (Figure 7).
1714
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
8- Different Amino Acids: The influence of different
eleven amino acids on polygalacturonase productivity
by the most potent bacterial strain viz. Bacillus firmusI-10104 was studied. The tested amino acids were
introduced as nitrogen sources, at equimolecular
amount. The aim of this experiment was to determine
the best amino acid that induces the highest enzyme
productivity. The results were revealed that all the
tested amino acids exhibited various degrees of
polygalacturonase activity lower than the control
(Figure 8).
9- Different Vitamins: All of the tested vitamins exert
suppressive effects on polygalacturonase productivity by
Bacillus firmus-I-10104 at concentrations 100, 250, 500
and 1000 ppm (Figure 9).
10- Different Aeration Conditions: As it is shown in
fig. (10) the 500 ml flask volume was more favorable
for polygalacturonase productivity, where it reached up
to 297.5 U/ml by Bacillus firmus-I-10104. Data
recorded
in table (3) showed a summary of the
optimal nutritional and environmental conditions for
Fig 6: Relation among polygalacturonase productivity by Bacillus firmus-I-10104 with different nitrogen sources
under solid state fermentation conditions.
Fig 7: Effect of different carbon sources on the polygalacturonase productivity using Solanum tuberosum (ST) peels
under solid state fermentation conditions by Bacillus firmus-I-10104.
1715
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
Fig 8: Effect of different amino acids on the polygalacturonase productivity using Solanum tuberosum (ST)
peels under solid state fermentation conditions by Bacillus firmus-I-10104.
polygalacturonase production by Bacillus firmus-I10104 grown on potato peels wastes as preferable
substrate.
Discussion Enzyme production is a growing field of
biotechnology and the world marked for enzyme is
over 1.5 billion and it is anticipated to double by the
year 2008 [3 0 ]. The majority of the industrial enzymes
are of microbial origin [4 5 ]. In the present study, fifty
one bacterial isolates were isolated from potato peels
collected from different restaurants in Benha, Kalubeia
governorate, Egypt. These bacterial isolates were grown
at 37 °C and at pH 6.2 to be able to produce a
polygalacturonase which favorable to be used as
additive for clarification of the juices. A screening of
pectinolytic productivities of the 51 bacterial isolates
showed that, twenty bacterial isolates gave a good
pectinolytic productivities. The nature of solid substrate
is the most important factor in solid state fermentation
(SSF). This not only supplies the nutrients to the
culture but also serves as an anchorage for the growth
of microbial cells [4 8 ]. The selection of substrate for
SSF process depends upon several factors mainly
related with the cost of availability and this may
involve the screening of several agro-industrial
residues. An optimum solid substrate provides all
necessary nutrients to the microorganism for optimum
function. However, some of the nutrients may be
available in sub-optimal concentrations or even not
present in the substrates. In such cases, it would be
necessary to supplement them externally [4 8 ]. Indeed 3040 % of the production cost for industrial enzymes are
accounted for the cost of the culture medium. In order
to reduce medium costs we screen different low-cost
substrates and in the course of this we identified potato
peels for cost-effective production of the enzyme under
study. SSF is receiving a renewed surge of interest,
primarily because increased productivity and prospect
of using a wide agro-industrial residues as substrates
. From industrial point of view, in order to achieve
production of low cost of enzymes these bacterial
isolates under study were allowed to grow on natural
substances such as Solanum tuberosum (ST) peels,
Solanum melanogena (SM) peels, Echronia crasipes
(EC) and citrus peels mixture (CPM) under solid state
fermentation (SSF). However, the selection of the
previously mentioned substrates for the process of
enzymes biosynthesis was based on the following
factors viz (i) they represent the most cheapest agroindustrial wastes in Egypt; (ii) they are available at any
time of the year; (iii) their storage represents no
problem in comparison with other substrates and (iv)
they resist any drastic effect due to the exposure to
other environmental conditions e.g. temperature,
variation in the weather from season to season and/or
from day to night. SSF are usually simpler and can use
wastes of agro-industrial substrates for enzyme
production. The minimal amount of water allows the
production of metabolites in a more concentrate from
making the downstream processing less time consuming
and less expensive [3 5 ,1 4 ]. Higher production 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 allowing them to utilize the substrate
effectively [3 4 ]. This trial appeared that only three
bacterial isolates B-10104, B-107 and B-4071 were
considered to be the best for pectinases production by
growing on ST peels under solid state fermentation
(SSF) conditions. They were identified as Bacillus
firmus-I-10104, Bacillus laterosporons-I-107 and
Bacillus firmus-I-4071. These results agree with that
obtained by Kapoor et al., [2 3 ] who reported that, the
members of the genus Bacillus and related genera are
known to produce extracellular pectinases, which have
applications in fiber industry. Bayoumi [6 ] produced
[3 6 ]
1716
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
polygalacturonase from Bacillus cereus. Kobayashi et
al. [2 7 ] purified the first bacterial Exo-PG from Bacillus
sp. strain KSM-P443. In the present study, Bacillus
firmus-I-10104 was a Gram-positive rod, catalase
positive, spore forming bacteria and grew in both
aerobic and anaerobic conditions. These results are
connected with that recorded by Kapoor et al., [2 2 ] who
found that, Bacillus sp. MG-CP-2 produce an alkaline
and thermostable PG in degumming of ramie
(Boehmeria nivea) and Sunn hemp (Crotalaria juncea).
The environmental conditions in solid-state fermentation
conditions can stimulate the microbe to produce the
extracellular enzymes with different properties other
than those of enzymes produced by the same organism
under the conditions performed in submerged
fermentation [3 4 ]. In this field, many workers dealt with
the main different factors that affect the enzymes
production such as temperature, pH, aeration, addition
of different carbon and nitrogen sources. Although such
factors were previously studied by many authors, [2 9 ].
Still, we need for more investigation seems to be
continuously required to give a chance to isolate more
Fig 9: Relation between polygalacturonase productivity by Bacillus firmus-I-10104 with different vitamins
concentrations under solid state fermentation (SSF) conditions.
Fig 10: Effect of different flask volumes on the polygalacturonase productivity using Solanum tuberosum (ST)
peels under solid state fermentation conditions by Bacillus fimus-I-10104.
1717
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
Table 3:
N o.
1
2
3
4
5
6
7
8
9
10
A sum m ary the optim al nutritional and environm ental param eters controlling polygalacturonase productivity by Bacillus firm us-I10104 under solid state ferm entation conditions.
Param eter
Bacillus firmus-I-10104
Inoculum size (m l)
1 ml
Substrate concentration (gm /25 m l)
1.25
Incubation period (hours)
96
pH value
6.2
Tem perature ( °C)
37
N itrogen source
Peptone
Carbon source
Control
Am ino acids
Control
Vitam ins (100, 250, 500 and 1000 ppm )
Control
Flask volum e (m l)
500
the present work is to determine the optimum
conditions for the enzyme (s) productivities by Bacillus
firmus-I-10104. On the other hand, the economic
feasibility of the microbial enzymes production
application generally depends on the cost of its
production processes. In order to obtain high and
commercially viable yields of pectinases enzymes, it
was essential to optimize the fermentation medium
used for bacterial growth and enzymes production.
Optimal parameters of the pectinases enzymes
biosynthesis from microbial origin, varied greatly, with
the variation of the producing strain, environmental,
and nutritional conditions [4 4 ]. Concerning the effect of
inoculum size, the obtained results revealed that, the
optimal inoculum size needed to produce the highest
yield of polygalacturonase (PG) production were 1.0
ml/ 25 ml in flask (250 ml). Each one ml of the heavy
bacterial cell suspension contained about 30 x 10 1 5
CFU. Inoculum size above the previously optimum
recorded value gave value gradually decreasing as
compared to that of the optimal one. This may be due
to the amount of ST peels was limited (1.25 g/ 25 ml)
in flask 250 ml and any increasing in inoculum size
does not producing any increasing in the yield of PG.
In this respect, Kapoor et al., [2 2 ] found that, production
medium (50 ml) in a 250 ml Erlenmeyer flask was
inoculated with 0.1 % of 12h old seed culture of
Bacillus sp. M G-CP-2 and incubated at 30 °C with
shaking (200 rpm). On the other hand, Kaur et al., [2 6 ]
found that, citrus peel-yeast extract broth was
inoculated with 20 x 10 7 conidiospores per 50 ml
medium from a 6-day old culture of Sporotrichum
thermophile Apinis. The differences may be due to
they inoculated S. thermophile Apinis but we
inoculated B. firmus-I-10104. Also, Kashyap et al. [2 5 ]
found that, 2.0 ml of culture of Bacillus sp. DT7
growing on wheat bran under SSF was used an
inoculum in each 250 ml Erlenmeyer flask. Concerning
the effect of substrate concentrations, the obtained
results revealed that, the maximum PG productivity
437.5 U/ml was obtained in presence of 1.25 g/ 25 ml
on ST peels. Substrate concentrations below and above
this value gave value gradually decreasing as compared
to that of the optimal one. This may be due to the
inolucum size was limited [1.0 ml/ flask (250 ml)] and
any increasing in substrate concentration doesn't
producing any increasing in the PG yield. In this field,
Zheng and Shetty [5 2 ] stated that, Erlenmeyer flasks
(125 ml), containing 10 g of dried apple or cranberry
of strawberry pomace and 20 ml of water and covered
with cotton plugs were autoclaved at 121 °C for 15
min. The Lentinus edods mycelium from one PDA
plate was inoculated into ten flasks of each substrate
separately. The flasks were incubated at 25 °C for 50
days. The cultivation of the fungus was also
extrapolated for 100 g of fruit pomace with
proportional addition of water calculated from the 10
g level. Also, Kashyap et al., [2 5 ] found that, solid
substrates (5.0 g; wheat bran, rice bran, apple pomace)
in 250 ml Erlenmeyer flasks were moistened with
specified content of distilled water for growing Bacillus
sp. DT7 which produced pectinase under SSF. In the
present study, incubation period has an obvious effect
polygalacturonase by Bacillus firmus-I-10104, it seems
from the results that a lag phase was observed during
the first 24h when spore germination took place with
practically no enzyme synthesis. M aximal PG
productivity on ST peels was observed at the end of
98 h, after which a decline in enzyme activity was
observed. This might be due to denaturation and/or
decomposition of PG as a result of interaction with
other compounds in the fermented medium [3 8 ] or due
to sugar consumption [1 6 ]. In accordance to the present
results, Martins et al., [3 2 ] found that, PG production
peaked between the 2 n d and 4 th days of cultivation
when wheat bran or orange bagasse was used as
substrate for Thermoascus aurantiacus. On the other
hand, different incubation periods were recorded by any
authors for Aspergillus foetidus e.g. 36h [1 6 ] might be
due to who use Aspergillus foetidus growing on citrus
waste but we use Bacillus firmus-I-10104 growing on
ST peels. The highest PG activity was obtained after
40 days by Lentinus edodes growing on straw berry
pomace medium under SSF conditions [5 2 ]. These
results are related with those recorded by Kaur et al.,
[2 6 ]
who reported that, PG production by the
thermophilic mould S. thermophile Apinis was high
after 4 days in SmF. As regarded the effect of pH on
PG production the present results showed that the
optimal pH for highest yield of the PG enzyme from
B. firmus-I-10104 was 6.2. Below and above this
optimal pH, the enzyme productivity decreased
gradually. This might be due to the growth rate of the
B. firmus-I-10104 was decreased above this pH and the
1718
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
acidity and alkalinity was harmful for bacteria. These
results agree with that obtained by Zheng and shetty,
[5 2 ]
who reported that, PG produced from Lentinus
edodes has a relatively lower pH optimum (pH 5.0).
Kobayashi et al., [2 7 ] reported that, PG produced by
Bacillus sp. Strain KSM-P443 growing on a pectate
agar plate in 100 mM tris-HCl buffer (pH 7.0). In
accordance to the present results, Debing et al., [1 3 ]
found that the optimal formula for pectinase production
from A. niger by solid fermentation under the
conditions of natural aeration, natural substrate pH
(about 6.5), and environmental humidity of 60 % is
rice dextrose 8%, wheat bran 24%, ammonium sulfate
[(NH 4 ) 2 SO 4 ] 6%, and water 61% , T ween 80 was found
to have a negative effect on the production of pectinase
in solid substrate. The present results showed that, the
PG optimal temperature of the enzyme were 37 °C.
Below and above this optimal temperature, the enzyme
productivity decreased gradually. This might be due to
the fact that lower and higher temperature cause a
decrease in the growth rate of B. firmus-I-10104 and
also below, this temperature the bacteria can not
perform its metabolic activities and above this
temperature the PG was broken down. In complete
accordance with the present results, Kashyap et al. [2 5 ]
found that, Bacillus sp. DT7 growing on wheat bran
under SSF yielded 4600 V of pectinase at 75%
moisture level after 36 h of incubation at 37 °C for
specified time intervals. On the other hand, Kaur et al.,
[2 ]
found that, the production of PG by Sporotrichum
thermophile Apinis in stirred SmF was high in
comparison with that instatic conditions. Yeast extract
(0.25%) and citrus pectin (2%) at pH 7.0 and 45 °C
supported a high enzyme production in flasks agitated
at 200 rpm. Tseng et al., [5 0 ] found that, B. firmus
which isolated from a wastewater treatment plant of
pulp and paper industry was capable of growth at pH
values, ranging from 10-12. Normally growth is at
temperature of 37 °C, the cultures were thermobile at
temperature above 55 °C. Concerning the effect of the
addition of different nitrogen sources for the purpose of
biosynthesis and production of PG, it was found that,
peptone, gelatin and ammonium chloride were the best
PG inducers for B. firmus-I-10104. This might be due
to peptone and gelatin are natural nitrogen sources. In
this respect, Kashyap et al., [2 5 ] found that, 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%. Addition of glycine, urea and
ammonium nitrate inhibited pectinase production, 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. In
a trial to study the effect of introducing some carbon
sources on the polygalacturonase production, it was
found that, all the tested carbon sources failed to
induce polygalacturonase production by growing
Bacillus firmus-I-10104 on Solanum tuberosum (ST)
peels under SSF conditions. This can be attributed to
ST peel, which has a high pectin content, and could
p r o d u c e s r e d u c in g suga rs b y a tta c k in g b y
polygalacturonase during fermentation. These results are
related with that given by Kapoor et al., [2 2 ] who
reported that, in minimal medium supplemented with
1% (w/v) citrus pectin as the sole carbon source, the
maximum polygalacturonase yield obtained was 47
U/ml after 24h, by using Bacillus sp. MG-CP-2 under
SmF and also, found that, polygalacturonase production
by Bacillus sp. M G-CP-2 using various sugars and
c o m p le x a gro-by p rod uc ts s how ed that the
polygalacturonase production was enhanced to a
significant extent using 1 % (w/v) wheat bran (189
u/ml), sun flower seed cake (144 U/ml), rice bran (141
U/ml), orange peel and guar gum (140 U/ml, each)
when substituted individually in place of citrus pectin
in the enriched medium. Maltose, lactose, sucrose and
mannitol did not show any significant effect on
polygalacturonase production, whereas rhamnose,
arabinose, glucose and galactose marginally inhibited
the enzyme production. Inhibition of polygalacturonase
production in the presence of glucose and other simple
sugars might be due to catabolite repression. Similar
results showing a higher pectinase yield with pectin
polymer compared with simple sugars such as
arabinose, glucose and galactose has been reported
earlier by Said et al., [4 1 ] which is in accordance with
the present study. Beg et al., [7 ,8 ] found that, pectinase
production from Streptomyces sp. QG-11-3 was
enhanced by wheat bran and drastically inhibited by
various mono- and disaccharides. The overall study
indicated that the complex polysaccharides such as ST
peels are the better stimulant for the polygalacturonase
production from Bacillus firmus-I-10104 compared with
simple sugars. Concerning the effect of aeration
conditions, the obtained results revealed that, 500 ml
flask volume was more favorable for PG productivity,
where it reached up to 297.5 U/ml. This may be due
to 500 ml flask volume supply B. firmus-I-10104 by
enough aeration needed for respiration. Then the
bacteria can perform its metabolic activities in a good
way. In this respect, Kaur et al., [2 6 ] found that, 250 ml
Erlenmeyer flask was more favorable for PG
production by Sporotrichum thermophile Apinis under
SmF. Moreover, some other workers [5 2 ] recorded that,
Erlenmeyer flask (125 ml) was the best for PG
production by Lentinus edodes under SSF. Sharma and
Satyanarayana [4 6 ] reported that, the pectinase
production further increased 41 fold, when enzyme
production was carried out in a fermentor, this could
be attributed to uniform distribution of nutrients and
improved aeration. Improvement in product yield is
expected in the fermentor as compared to that in flasks,
because of better control of process parameters in the
former [2 1 ]. M aximum pectinase production in the
fermentor was attained in 30 h as compared to that of
40 h in shake flasks. A similar reduction in
fermentation time was recorded in the production of a-
1719
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
amylase by G. thermoleovorans, where the optimum
production was achieved in 7 h in fermenter as
compared to 12 h in shake flasks [3 9 ].
REFERENCES
1.
Agrios, G.N., 1988. How pathogens attack plants.
Ch.3 In: Plant pathology 3 rd ed., (pp. 63-86).
Academic Press, Inc., San Diego, C.A.
2. Ahlawat, S., B. Battan, S.S. Dhiman, J. Sharma
and R.P. M andhan, 2007. Pro d uction of
thermostable pectinase and xylanase for their
potential application in bleaching of kraft pulp.
J.Ind.Microbiol Biotechnol., 34: 763-770.
3. Babu, K.R. and T . Satyanarayana, 1995. α-amylase
production by the thermophilic Bacillus coagulans
in solid state fermentation. Process B iochem., 30:
305-309.
4. Bai, Z.H., H.X. Zhang, H.Y. Qi, X.W . Peng and
B.J. Li, 2004. Pectinase production by Aspergillus
niger using
wastewater
In
solid
state
fermentation
for
eliciting
plant
disease
resistance. Bioresource Technol., 95: 49-52.
5. Barrow, G.I. and R.K. Feltham, (Eds.), 1993.
Cowan & Steel's Manual for the Identification of
Medical Bacteria 2 n d ed. Cambridge Univ. Press
London.
6. Bayoumi, R.A., 1997. Environmental studies on
microflora of certain Egyptian antiquities objects,
M.Sc. Thesis, B ot. and Microbiol. Dept., Fac. of
Sci., Al-Azhar Univ.
7. Beg Q.K., B. Bhushan, M. Kapoor and G.S.
Hoondal, 2000a. Enhanced production of a
thermostable xylanase from Streptomyces sp. QG11-3 and its application in biobelaching of
eucalyptus kraft pulp Enzyme Micorb Technol
2000, 27: 459-66.
8. Beg Q.K., B. Bhushan, M. Kapoor, G.S. Hoondal,
2000b. Production and characterization of
thermostable xylanase and pectinase from
Streptomyces sp. QG-11-3 J Ind Microbiol
Biotechnol 2000, 24: 396-402.
9. Bohdziewiez, J. and M. Bodzek, 1994.
Ultrafiltration preparation of pectinolytic enzymes
from citric acid fermentation broth. Proc.
Biochem., 29: 99-107.
10. Castilho, L.R., T.L.M. Alves and R.A. M edronho,
1999a. Recovery of pectinolytic enzymes produced
by solid state culture of Aspergillus niger, Process
Biochem., 34: 181-186.
11. Castilho, L.R., R.A. Medronho and T.L.M. Alves,
1999b. Production and extraction of pectinases
o b ta ine d b y so lid state ferm en ta tion of
agroindustrial residues with Aspergillus niger.
Bioresource Technol., 71: 45-50.
12. Collee, J.G., B.P. Marmion, A.G. Fraser and A.
Simmons, 1996. Medical Microbiology (Mackie
and Mac Carteny). Froteen Edition. Chruchill
Livingstone.
13. Debing, J., L. Peizun, F. Stagnitti, X. Xianzhe and
L. Li, 2005. Pectinase production by solid
fermentation from Aspergillus niger by a new
prescription experiment. Ectoxicology and Environ.
safety, 64: 244-250.
14. El-Sheekh, M ., A.S. Ismail, M .A. El-Abd, E.M.
Hegazy and A. El-Diwany, 2008. Effective
technological pectinases by Aspergillus carneus
NRC1 utilizing the Egyptian orange juice industry
scraps. Inter. Biodeterior. & Biodegrad. 1-7. under
publication.
15. Favela-Torres, E., T.V. Sepulveda and ViniegraG onzalez, 2006. P roduction of hydrolytic
depolymerising pectinases. Biotechnol., 44(2): 221227.
16. Garzon, C.G. and R.A. Hours, 1992. Citrus waste:
Alternative substrate for pectinase production in
solid-state culture. Bioresource technology, 39: 9395.
17. Gessesse, A. and G. Mamo, 1999. High level
xylanase production by an alkalophilic Bacillus sp.
by using solid state fermentation. Enzyme Microb.
Technol., 25: 68-72.
18. Goodman, R.N., Z. Kiraly and K.R. W ood, 1986.
Cell wall composition and metabolism. Ch.4 In:
The Biochem. and Physiol. of plant Disease,
(pp.105-149), Univ. of Missouri Press, Columbia.
19. Gummadi, S.N. and T. Panda, 2002. Purification
and biochemical properties of microbial pectinases:
a review Process Biochem., 38: 987-996.
20. Hensyl, W .R. (Ed), 1994. B ergy's Manual of
Determenative Bacteriology 9 th edition, W illiams &
W ilkins, Baltimore.
21. Humphrey, A., 1998. Shake flask to fermenter,
what have we learnt? Biotechnol. Prog., 14: 3-7.
22. Kapoor, M., Q.K. Beg, B. Bhushan, K.S. Dadhich
and G.S. Hoondal, 2000. Production and partial
purification and characterization of a thermo-alkali
stable polygalacturonase from Bacillus sp. MG-cp2. Process Biochem., 36: 467-473.
23. Kapoor, M., Q.K. Beg, B. Bhushan, K. Singh,
K.S. Dadhich and G.S. Hoondal, 2001. Application
of an alkaline and thermostable polygalacturonase
from Bacillus sp. MG-cp-2 in degumming of ramie
(Bochmeria nivea) and sunn hemp (Crotalaria
juncea) bast fibers, Process Biochem., 36: 803807.
24. Kashyap, D.R., P.K. Voha, S. Chopra and R.
Tewari, 2001. Applications of pectinases in the
commercial sector: A review Bioresou. Technol.,
77: 216-285.
25. Kashyap, D.R., S.K. Soni and R. T ewari, 2003.
Enhanced production of pectinase by Bacillus sp.
DT7 using solid state fermentation. Bioresou.
Technol., 88: 51-254.
26. Kaur, G., S. Kumar, and T. Satyanarayana, 2003.
Production, characterization and application of a
thermostable plygalacturonase of a thermophilic
mould Sporotrichum thermophile Apinis. Bioresou.
Technol., 94: 239-243.
1720
J. Appl. Sci. Res., 4(12): 1708-1721, 2008
27. Kobayashi, T., N. Higaki, N. Yajima, A.
Suzumatsu, H. Hagihara, S. Kawai and S. Ito,
2000. Purification and properties of a Galacturonic
Acid-Releasing Exopolygal-acturonase from a
strain of Bacillus. Biosci. Biotechnol. Biochem.,
65(4): 842-847.
28. Lang, C. and H. Dornenburg, 2000. Perspectives
in the biological function and the technological
application of polygalacturonaes. Appl. Microbiol.
Biotechnol., 53: 366-375.
29. Lee, D., Y. Kok, K. Kim, B. Kim, H. Choi, D.
Kim, M.T. Suhartono and Y. Pyun, 1999. Isolation
and characterization of a thermophilic lipase
from Bacillus thermoleovorans ID-1 FEM S.
Microbiol. Lett., 179: 393-400.
30. Lowe, D.A., 2002. Production of enzymes In:
Basic biotechnology, Ralledge, C. and Kristiansen,
B. (eds.) 2 n d edition, Cambridge, pp. 391-408.
31. Lowry, O.H., N.G. Rosebrough, A.L. Farr and R.J.
Randall, 1951. Protein measurement with the folin
phenol reagent J. Bio. Chem., 193: 265-275.
32. Martins, E.S., D. Silva, R. Da Silva and E.
G omes, 2002. S o lid state p rod uction of
therm o stab le pectinases fro m therm ophilic
Thermoascus aurantiacus. Process Biochem., 37,
949-954.
33. Nelson, N., 1944. A photometric adaptation of the
somogyi Method for the Determination of Glucose,
J. Biol. Chem., 153: 375.
34. Pandey, A., C.R. Soccol, P. Nigam and V.T.
Soccol, 2000. Biotechnological potential of agroindustrial residues I: Sugar cane bagasse3 Biores
Technol., 74: 69-80.
35. Pandey, A., G. Szakaes, C. Soccol, J. RodriguezLeon and V. Soccol, 2001. Production, purification
and properties of microbial phytases, Bioresou.
Technol., 77: 203-214.
36. Park, Y.S., S.W . Kang, J.S. Lee, S.I. Hong and
S.W . Kim, 2002. Xylanase production in solid
state fermentation by Aspergillus niger mutant
using statistical experimental design. Appl.
Microbiol. Biotechnol., 58: 761-766.
37. Parker, M .T. (ed.), 1984. Principles of
Bacteriology, Virology and Immunity. Toply and
W ilson's. Seventh edition. Volume . Systematic
Bacteriol.
38. Ramesh, M.V. and R.K. Lonsane, 1987. Solid
state fermentation for production of a-amylase by
Bacillus megaterium 16M Biotechnol. Lett., 9:
323-328.
39. Rao, J.L.U.M. and T . Satyanarayana, 2003.
Statistical optimization of a high maltose forming,
hyperthermostable and Ca 2 + -independent a-amylase
production by an extreme thermophile Geobacillus
thermoleovorans using response surface methodol.
J. Appl Microbiol., 95: 712-718.
40. Reid, I. and M . Ricard, 2000. Pectinase in paper
making solving retention problems in mechanical
pulps bleached with hydrogen peroxide. Enzyme
Microbiol. Technol., 26: 115-123.
41. Said, S., M .J.V. Fonseca and V. Siessere, 1991.
Pectinase production by Penicillium frequentans.
W orld. J. Microbiol. Biotechnol., 7: 607-608.
42. Sakai, T., T. Sakamoto, J. Haellert and E.
V a nd am m e 1993a. P ectin, P ectinase and
protop ectinase: pro duction, pro perties and
applications. Adv. Appl. Microbiol., 25: 213-294.
43. Schallmey, M ., A. Singh and W .P. W ard, 2004.
Developments in the use of Bacillus species for
industrial production. Can. J. microbial., 50; 1-17.
44. Sharaf, E.F. and M.S. Ammar, 2000. Purification
and characterization of constitutive thermostable
three isoproteases produced by Humicola grisea
Var Thermodeae at 55 °C. Egypt J. Biotechnol., 8:
124-189.
45. Sharma, R., Y . Chisti, and U.C. Banerjee, 2001.
Production purification, characterization, and
applications of lipases, Biotechnol. Advances, 19:
627-662.
46. Sharma, D.C. and T. Satyanarayana, 2005. A
marked enhancement in the production of a highly
alkaline and thermostable pectinase by Bacillus
pumilus decr1 in submerged fermentation by using
statistical methods. Bioresou. Technol., 97: 727733.
47. Silva, D., K. Tokuioshi, E.D. Martins, R.D. Silva
and E. Gomes, 2005. Production of pectinase by
solid sta te fer m e ntatio n w ith P e nicillum
viridicatum RFC3. Process Biochemistry. 40:
2885-2889.
48. Sneath, P.H. (Editor), 1986. Bergey’s Manual of
systematic Bacteriology (Volume 2), W illiams &
W ilkins.Baltimore, London, Los -Angeles, Sydney.
49. Sodhi, H ., K . Sharma, J. Gupta and S. Soni, 2005.
Production of a thermostable a-amylase from
Bacillus sp. PS-7 by solid state fermentation and
its synergistic use in the hydrolysis of malt starch
for alcohol production. Process Biochem., 40: 525534.
50. T se ng,
M in-J e n,
Y ap,
M e e -N agan,
K.
Ratanakhanokehai, K.L. Kyu and Chen Shui-Tein,
2001. Purification and characterization of two
cellulase free Xylanases from an alkaliphilic
Bacillus firmus, Enzyme and Microbial Technol.,
30: 59-595.
51. Vincent, J.M., 1970. A M anual For The Practical
Study of The Root Nodule Bacteria. International
Biological Programme 7 Marylebone Rood,
London, NW i, Blackwell Scientific Publications,
Oxford & Edinburgh, pp.75.
52. Zheng, Z. and K. Shetty, 1999. Solid state
production of polygalacturonase by Lentinus
edodes using fruit processing wastes. Process
Biochem., 35: 825-830.
1721