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
1Reda, A. Bayoumi, 2Hesham, M.Yassin, 2Mahmoud, A. Swelim, 2Ebtsam,Z.Abdel-All
1Botany & Microbiology Department, Faculty of Science, Al-Azhar University,Cairo,Egypt.P.O.11884.
2Botany 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 agro-
industrial 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 1015 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(P G )production, Bacillus firmus, Solid State Fermentation
(SSF),
Agro-Industrial wastes
INTRODUCTION
by breaking glycosidic bonds of the long carbon chains
(polygalacturonase, pectin lyase and pectate lyase) and
M ost of the chemical changes that occur in living
by splitting off methoxyl groups (pectin esterase)
tissues are regulated by enzymes. In recent years there
[1 0 ,1 1 ,1 2 ;1 9 ;4 ]. Pectic substances are widely distributed in
has been a renewed interest in solid-state fermentation
fruits and vegetables [10-30 %] in turnips, peels of orange
(SSF) processes for the production of bioactive
and in pulps of tomato, pineapple and lemon), hence
compounds. Enzymes production by SSF using
they form important natural substrates for pectinases
bacterial spp. has been reported for many enzymes
[1 9 ]. Enzymes which degrade pectic substances are
such as xylanase [17] and amylase [3] but reports on
pectinases or pecteolytic enzymes and can be classified
pectinase production by SSF using bacterial spp. are
into three types. Pectin methyl esterase (PM E)
lacking in the literature. SSF has been reported to be
hydrolyzes the methyl ester of galacturonide chain
more advantageous than submerged (SmF) as it allows
liberating methanol. Polygalacturonases (PGases) and
c heap er p ro ductio n o f enzym e h a vin g b e tte r
pectate lyases (PLases) split the molecular chains of
physiochemical properties than that produced by
the respective polymers [18,1,24]. Since the 1940s,
SmF[25]. Pectinases comprises a heterogeneous group of
pectinases have been exploited for many industrial
enzymes that catalyze the breakdown of pectin-
applications. Pectinases are mainly used for increasing
c o n t a i n i n g su b s tr a te s . P e c tic s u b s ta n c e s a r e
filtration efficiency and clarification of fruit juices, in
characterized by long chains of galacturonic acid
wood preservation and used in maceration, liquefaction
residues. On these residues are carboxyl groups, which
and extraction of vegetable tissues [9,4]. Various
are sometimes modified by the addition of methyl
literature reports and reviews are available on the
groups, forming methoxyl groups. Pectic enzymes act
production and applications of pectinases [42,24]. Also,
Corresponding Author: Reda Ahmed Bayoumi, Bayoumi, R.A., Botany & Microbiology Department, Faculty of Science, Al-
Azhar University, Cairo, Egypt. P.O.11884.
Tel.: +(0020) 24108598;
Fax:+(0020) 22629356. Mobile: 0103597140
E-mail address: redaelbayoumi@yahoo.com
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J. Appl. Sci. Res., 4(12): 1708-1721, 2008
pectinases have been used in the paper and pulp
sterilization. The medium was poured into sterilized
industry in addition to cellulases [40], and plant
plates, each plate contained 20 ml, the media were
pathology [28]. Few reviews have highlighted the
allowed to be solidify.
biological and technological importance of pectinases[40,
2 4 ,1 5 ,2 ,1 4 ].
5. Agro-industrial W astes: The potato peels wastes
In this study, we report the nutritional and
were collected from different restaurants in Benha,
environmental conditions requirements for production
Kalubia governorate, Egypt. They washed to remove
of PG by Bacillus firmus-I-4071 under solid state
the clay for using, dried in open air, and then grounded
fermentation conditions using potato peels.
in the production media. Citrus peels, Solanum
tuberosum (ST) peels, and water hyacinth (Echornia
M ATERIALS AND M ETHODS
crassips) were collected, dried and prepared in the
form of a ground preparations.
1. Isolation of Bacteria: Bacterial isolates were
obtained from clayed potato peels collected from
6. Production M edia:
different restaurants in Benha, Kalubia governorate,
6.1. Basal M edium: The basal medium (BM ) was
Egypt. The potato peels were applied using the soil
prepared according to Vincent [51]. It contained of the
dilution plate method.
following (g/l): Sucrose, 10; KNO , 0.6; KH PO , 1;
3
2
4
M gSO , 0.25 and CaCl , 0.1 was found most
4
2
2. Construction of Galacturonic Acid Standard
convenient for the production of different enzymes. It
Curves: A stock solution (10.000 mg/ml) of the
was modified to include the following constituents:
standard galacturonic acid supplied by Sigma was
(g/l) NaNO , 2; K HPO , 0.5; KCl, 0.5 and yeast
3
2
4
prepare in acetate buffer (0.2 M ) at pH 5. The stock
extract, 1. These previously mentioned contents were
so lutio n wa s use d fo r m aking o f d i ffe r e n t
dissolved in citrate phosphate buffer at pH 7.
concentrations. After preparing of the required
dilutions, only 1.0 ml of each dilution was transferred
6.2 Potato Peels Basal M edium (PPM A): It contained
to determine of the amount of reducing sugar according
the same constituents of BM supplemented with potato
to N elson [33]. A standard curve was constructed
peels (4 % w/v).
relating all different sugar concentrations applied
against their corresponding to optical density at 510
7. Identification of the M ost Potent Bacterial
nm. The obtained standard curve was used for
Isolates: The three most potent bacterial isolates (B-
estimating the polygalacturonase activities in terms of
10104, B-107 and B-40710) were identified by
mg/ml and then units (U). One unit is defined as the
examination of their morphological physiological and
amount of enzyme protein (mg) required to exert free
biochemical characteristics according to Barrow and
galacturonic acid, from pectin of time under 35 °C for
Feltham [5]; Parker, [37]; Collee et al., ;
[1 2 ]
Sneath
1hour in acetate buffer (0.2 M ) at pH 5.0
[4 9 ];Hensyl, [20] and Schallmey et al., .
[4 3 ]
.
3. Protein Determination: Protein of all enzymatic
8.qualitative Screening Test M edia, M ethods, and
preparations was determined by the method of Low ry
Conditions (First Survey):
et al., [31].
8.1. Pectinolytic Enzyme Production M edium: This
medium consists of part (A) and part (B).
4. Grow th and M aintenance M edium: a-Czapek's-
Part (A) contained (g/l): NaNO , 2; KH PO , 1; KCl,
Dox pectin medium:
3
2
4
0.5; M gSO .7H O, 0.5; Yeast extract, 1. These contents
This medium contained (g/l): Pectin, 10; NaNO ,
4
2
3
were dissolved in 40 ml distilled water. The pH was
2; K H PO , 1; KCl, 0.5; M gSO .7H O, 0.5;
2
4
4
2
adjusted at pH 7 by NaOH (5% , w/v).Part (B)
FeSO .7H O, 0.001%, CaCl , 0.001%, agar-agar, 15;
4
2
2
contained (g/l): Pectin, 5, dissolved in 10 ml. of
and distilled water up to 1000 ml. pH was adjusted at
distilled water.The two parts (A) and (B) were
7, and then autoclaved for 20 minutes at 1.5
autoclaved for 20 minutes at 1.5 atmospheric pressure
atmospheric pressure. This medium was modified as
and mixed together after autoclaving. This medium was
: follow Part (A) contained (g/l): NaNO , 2; KH PO ,
3
2
4
inoculated with bacterial isolates. This medium was
1; KCl, 0.5; M gSO .7H O, 0.5; yeast extract, 1; agar-
4
2
incubated at 37 °C for 96 hours, then assayed for
agar, 20. This contents were dissolved in 200 ml
pectinolytic productivity and activity were tested in the
citrate-phosphate buffer at pH 7. Part (B) contained
pectinase assay medium.
(g/l): Pectin, 5; dissolved in 200 ml citrate phosphate
buffer at pH 7. Part (C) citrus peels extract which
8.2. Pectinase Production and Activity Assay
contained (g/l): Citrus peels, 125 g, the pH of the
extract was adjusted at 7 before sterilization. The three
M edium: This medium was contained (g/l): Pectin, 1;
parts (A), (B), and (C) were autoclaved for 20 minutes
Arabic gum, 5; agar-agar, 15, these contents dissolved
at 1.5 atmospheric pressure and mixed together after
in citrate phosphate buffer at pH 6. It was autoclaved
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J. Appl. Sci. Res., 4(12): 1708-1721, 2008
at 1.5 atmospheric pressure for 20 minutes. Plates of
10.3. Different Incubation Periods:The most potent
the same size were poured with equally amounts of
bacterial isolate was allowed to grow on the waste and
pectinase assay medium in each Petri dish. After
incubated for 6, 12, 24, 48, 72, 96, 120, 144, 168 and
cooling, three wells were made by sterilized cork porer
192 hours respectively.
in each plate. Each well was inoculated with 0.1 ml of

filtrate which prepared by the filtration of the broth
10.4. Different pH Values:The production medium for
which grown on pectinolytic enzyme production
most potent isolate were prepared as previously
medium by filter paper. These plates were inoculated
mentioned. The pH was adjusted at different pH values
at 37 °C for 24 hours. Then clearing zones of the
viz, [7.5, 8.5, 9.0] by using boric acid-borax buffer and (3,
medium after addition of Logule's iodine solution were
5, 5.5, 6, 6.2, 6.4, 6.6 and 7.0) by using citrate-
investigated and taken as criteria for determining the
phosphate buffer. Inoculation and incubation conditions
pectinolytic productivity
were carried as previously mentioned.
.9.a. Qualitative Screening Test M edia, M ethods and
10.5. Different Temperatures: The most potent
Conditions (Second Survey):
bacterial isolate was allowed to grow on the grounded
9.a.1. Pectinolytic Enzyme Production M edium
potato peels (PP) medium at different temperatures viz.,
(Pepm): This medium contained the main ingredients
10, 15, 30, 37, 40, 45, 55, 65 and 75 °C respectively
of BM supplemented with potato peels, Solanum
for 96h.
tuberosum (ST) peels, Echornia crassips and citrus
peels mixture (2% w/v) separately. The pH of this
10.6. Different Nitrogen Sources: Production medium
medium was adjusted at 7 by dissolve its contents in
was supplemented with different nitrogen sources at an
citrate-phosphate buffer (pH 7). It was autoclaved at
equimolecular amount of nitrogen that present in
1.5 atmospheric pressure for 20 minutes. T his medium
sodium nitrate (0.2% , w/v) in basal medium. Peptone,
was inoculated with bacterial isolates under study. This
gelatin, and casein were introduced as organic nitrogen
medium was inocubated at 37 °C for 96 hours, then
source at the level of 2 % and the control was devoid
assayed fo r pectin o lytic p ro d uctivity in the
from any nitrogen source. The applied nitrogen sources
polygalactunase assay medium as previously mentioned
were ammonium sulphate, ammonium molybdate,
ammonium chloride, ammonium oxalate, ammonium
.9.b.medium Used in Screening Test for Selecting
citrate, ammo nium tartrate, ammonium nitrate,
the M ost Potent Bacterial Isolates: This medium
diammonium hydrogen phosphate, potassium nitrate,
contained the main ingredients of PEPM . The
gelatin, peptone, casine and urea. All other factors
pectinolytic activity was detected by Nelson's technique
(temperature, pH, substrate concentration and carbon
[3 3 ].This mixture was incubated at 45 °C for 20
sources) were carried out as previously mentioned.
minutes, assay for reducing sugar by Nelson's technique
as galacturonic acid. This mixture was modified as
10.7. Different carbon sources: Different carbon
follow: This mixture was incubated at 35 °C for 1 hour
sources were introduced into the production medium at
.
an equimolecular amount located at 1% (w/v) glucose.
9.c. Analysis of Reducing Sugar by Nelson's
Parallel experiment was made with no sugar as a
Technique :
[3 3 ]
control. The carbon sources were represented by
10-parameters Controlling the Polygalacturonase
xylose, arabinose, glucose, galactose, mannose,
Productivities:
fructose, trehalose, lactose, maltose, sucrose, starch,
10.1. Different Inoculum Sizes: Different inoculum
cellulose and pectin. Starch, cellulose and pectin was
sizes of heavy spore suspension of the most potent
introduced at the level of 1% (w/v). In all cases, other
bacterial isolate (prepared by harvesting 5 slants in 100
previously mentioned optimal conditions were taken
ml sterile saline solution under aseptic conditions) are
into consideration.
used. The following inoculum sizes were applied viz.,
1, 2, 5, 10, 15, 20, and 24 ml per each flask (250 ml).
10.8. Different Amino Acids: The production medium
At the end of incubation periods, polygalacturonase
was used after applying all of the previously mentioned
productivity was determined for each flask after
optimal environmental and nutritional conditions for
incubation period as the previously mentioned.
polygalacturonase productivity by the most potent
bacterial isolate. The used amino acids were added at
10.2. Different Substrate Concentrations: Different
an equimolecular amount of nitrogen located in the best
concentrations of substrate (g/flask, 25 ml, w/v) were
inorganic nitrogen source for the enzyme productivity.
applied viz., 0.1, 0.3, 0.5, 0.75, 1.0, 1.25, 1.5, 2.0, 5.0,
This experiment was controlled by performing of
10 and 15). At the end of incubation period, enzyme
parallel one containing the original nitrogen source i.e.,
productivity were assayed.
sodium nitrate. The control of this experiment was
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devoid of any amino acid, and the supplemented amino
program to the species level. The morphological
acids were: alanine, histadine, phenyl alanine, aspartic
characteristics and stain reaction led to the fact that the
acid, glycine, lysine, cystine, tryptophan, methionine,
three bacterial isolates are suggestive to being
arginine and isoleucine. Inoculation, incubation
belonging to the genus Bacillus, gram positive aerobes
co nd itio n s, a n d m e a su re m e n t o f the enzym e
to facultative anaerobes. Consulting Bergey's M anual of
productivity were performed as previously mentioned.
systematic bacteriology [49], the three isolates are
belonging to species firmus, firmus, and laterosporus.
10.9. Different Vitamin Requirements: Different
They could be give the tentative name Bacillus firmus
vitamins viz, ascorbic acid, riboflavin, vitamin B6 and
I-4 0 7 1 , B a c illu s firm u s-I-1 0 1 0 4 and B acillu s
folic acid were added separately to the medium
laterosporons-I-107.
specialized for polygalacturonase production at 100,
250, 500, and 1000 ppm while the control was applied
Parameters Controlling the Polygalacturonase (PG )
free from any vitamin. Inoculation, incubation
Productivity:
conditions, and measurement of enzyme productivity
1- Different Inoculum Size: Different volumes of
were performed as previously mentioned.
bacterial spore suspension were used as an inocula
sizes to inoculate flasks (250 ml) containing the
10.10. Different Aeration Conditions: This experiment
inoculum size used were 1, 2, 5, 10, 20 and 24 ml.
was carried out to investigate the effect of different
Each one ml of the heavy bacterial cell suspension
aeration conditions on polygalacturonase productivity.
contained abut 30 x 1015 CFU. The optimal inoculum
It was performed by using four different volumes viz,
sizes need to produce the highest yield of
100, 250, 500 and 1000 ml for agricultural waste as
polygalacturonase were 1.0 ml. At this particular
substrate. Each flask contained 25 ml of the medium in
inoculum size, the highest yield of polygalacturonase
case of potato peels production m edium for
was attained with Solanum tuberosum (ST) peels as
polygalacturonase. The enzyme productivity was
350 U/ml. Inoculum size above the previously optimal
assayed as previously mentioned.
recorded value gave value gradually decreasing as
compared to that of the optimum one(Figure 1).
Results Fifty one bacterial isolates were isolated from

fermented Solanum tuberosum (ST) peels. These
2- Different Substrate Concentrations: The maximum
isolates were purified, and subjected to a screening in
polygalacturonase productivity 437.5 U/ml was obtained
order to examine their pectinolytic productivities on the
in presence of 1.25g /25 ml on Solanum tuberosum
basis of mean diameters of clearing zones (mm). The
(ST) peels by Bacillus firm us-I-10104 at 37 °C for 96h
fifty one bacterial isolates had a pectinolytic activity
(Figure 2).
while twenty isolates of them were considered to give
good producers. T he twenty isolates to attack some
3- Different Incubation Periods: Effect of different
agriculture, and industrial wastes under solid state
in c u b a t io n p e r i o d s o n th e p o lyg a la c tu r o n a s e
fermentation (SSF) T he three bacterial isolates numbers
productivity using Solanum tuberosum (ST) peels under
4071, 107 and 10104 out of the twenty isolates gave a
solid state fermentation conditions by Bacillus firmus-I-
higher pectinase productivity by attacking Solanum
10104 was tested at time intervals of 6, 12, 24, 48, 72,
tuberosum (ST) peels compared to other wastes and
96, 120, 144, 168 and 192 hours. The level of
other isolates, where it reached up to 3.2, 3.4 and
polygalacturonase increased gradually with increasing
3.4mm respectively. Bacterial isolates numbers 4071,
the incubation period up to a maximum of 96h. Then
107 and 10104 also gave a higher polygalacturonase
gradually decreased after these periods (Figure 3).
productivity by attacking Solanum tuberosum (ST)
peels compared to other wastes and other isolates,
4- Different Initial PH Values: The polygalacturonase
where it reached up to 515, 515 and 367.5 (U/ml)
productivity by Bacillus firmus-I-10104 reached its
respectively. Bacterial isolates number 4071, 107 and
maximum at initial pH 6.0 and 6.2. Since the enzyme
10104 gave a higher polygalacturonase productivity by
yield reached up to 325 U/ml., below and above this
attack Solanum tuberosum (ST) peels compared to
optimal pH value, the enzyme productivity gradually
other wastes, where it reached up to 292, 287 and 297
decreased (Figure 4).
(U/ml) respectively (Tables 1&2).
5- D ifferen t Incu b a tio n T em p era tu res: T he
Identification of the Three M ost Potent Bacterial
polygalacturonase productivity reached its optimal value
Isolates: The three most potent bacterial isolates 4071,
310 U/ml at an incubation temperature at 37 °C
10104 and 107 were subjected to an identification
(Figure 5).
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T able 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 N elson's technique (33) (Second Survey).
N o.
C ode no. of bacterial isolates
Polygalacturonase productivity (U /m l)
--------------------------------------------------------------------------------------------------------------------------------
C itrus peels
Solanum tuberosum Solam um m elongena Echornia
m ixture (C PM )
(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
T able 2:
Q uan titative 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).
N o.
C ode no. of bacterial isolates
Polygalacturonase productivities (U /m l)
-----------------------------------------------------------------------------------------------------------------------------
Citrus peels m ixture (CPM )
Solanum tuberosum (ST) peels
Solanum m elanogena (SM ) peels
1
107
105 ± 0.05
287.5 ± 0.02
75 ± 0.01
2
4071
95 ± 0.09
292.5 ± 0
40 ± 0.04
3
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.
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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.
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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
7- Different C arbon Sources: The effect of thirteen
organic as well as inorganic nitrogen sources on
different carbon sources were introduced into the
polygalacturonase productivity by the most potent
applied production medium of polygalacturonase
bacterial strain Bacillus firmus-I-10104 were studied.
productivity by Bacillus firmus-I-10104 under SSF
Fourteen different nitrogen sources were applied as
condition were studied. It was clear that all the
equimolecular amount located in sodium nitrate. The
different carbon sources exhibited various degrees
maximum value of polygalacturonase productivity
lower than control by the Bacillus firmus-I-10104.
reached up to 350 U/ml in the presence of peptone
Solanum tuberosum (ST) peels was the best carbon
followed by ammonium chloride, urea and beef extract
source for polygalacturonase production where the
(Figure 6).
productivity reached up to 115 U/ml (Figure 7).
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8- Different Amino Acids: The influence of different
9- Different Vitamins: All of the tested vitamins exert
eleven amino acids on polygalacturonase productivity
suppressive effects on polygalacturonase productivity by
by the most potent bacterial strain viz. Bacillus firm us-
Bacillus firmus-I-10104 at concentrations 100, 250, 500
I-10104 was studied. The tested amino acids were
and 1000 ppm (Figure 9).
introduced as nitrogen sources, at equimolecular
amount. The aim of this experiment was to determine
10- Different Aeration Conditions: As it is shown in
the best amino acid that induces the highest enzyme
fig. (10) the 500 ml flask volume was more favorable
productivity. The results were revealed that all the
for polygalacturonase productivity, where it reached up
tested amino acids exhibited various degrees of
to 297.5 U/ml by Bacillus firmus-I-10104. Data
polygalacturonase activity lower than the control
recorded in table (3) showed a summary of the
(Figure 8).
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.
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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-I-
of using a wide agro-industrial residues as substrates
10104 grown on potato peels wastes as preferable
[3 6 ]. From industrial point of view, in order to achieve
substrate.
production of low cost of enzymes these bacterial
isolates under study were allowed to grow on natural
Discussion Enzyme production is a growing field of
substances such as Solanum tuberosum (ST) peels,
biotechnology and the world marked for enzyme is
Solanum melanogena (SM ) peels, Echronia crasipes
over 1.5 billion and it is anticipated to double by the
(EC) and citrus peels mixture (CPM ) under solid state
year 2008 [30]. The majority of the industrial enzymes
fermentation (SSF). However, the selection of the
are of microbial origin [45]. In the present study, fifty
previously mentioned substrates for the process of
one bacterial isolates were isolated from potato peels
enzymes biosynthesis was based on the following
collected from different restaurants in Benha, Kalubeia
factors viz (i) they represent the most cheapest agro-
governorate, Egypt. These bacterial isolates were grown
industrial wastes in Egypt; (ii) they are available at any
at 37 °C and at pH 6.2 to be able to produce a
time of the year; (iii) their storage represents no
polygalacturonase which favorable to be used as
problem in comparison with other substrates and (iv)
additive for clarification of the juices. A screening of
they resist any drastic effect due to the exposure to
pectinolytic productivities of the 51 bacterial isolates
other environmental conditions e.g. temperature,
showed that, twenty bacterial isolates gave a good
variation in the weather from season to season and/or
pectinolytic productivities. The nature of solid substrate
from day to night. SSF are usually simpler and can use
is the most important factor in solid state fermentation
wastes of agro-industrial substrates for enzyme
(SSF). This not only supplies the nutrients to the
production. The minimal amount of water allows the
culture but also serves as an anchorage for the growth
production of metabolites in a more concentrate from
of microbial cells [48]. The selection of substrate for
making the downstream processing less time consuming
SSF process depends upon several factors mainly
and less expensive [35,14]. Higher production of pectinase
related with the cost of availability and this may
in SSF process may be due to the reason that solid
involve the screening of several agro-industrial
substrate not only supplies the nutrient to the microbial
residues. An optimum solid substrate provides all
cultures growing in it, but also serves as anchorage for
necessary nutrients to the microorganism for optimum
the cells allowing them to utilize the substrate
function. However, some of the nutrients may be
effectively [34]. This trial appeared that only three
available in sub-optimal concentrations or even not
bacterial isolates B-10104, B-107 and B-4071 were
present in the substrates. In such cases, it would be
considered to be the best for pectinases production by
necessary to supplement them externally [48]. Indeed 30-
growing on ST peels under solid state fermentation
40 % of the production cost for industrial enzymes are
(SSF) conditions. They were identified as Bacillus
accounted for the cost of the culture medium. In order
firmus-I-10104, Bacillus laterosporons-I-107 and
to reduce medium costs we screen different low-cost
Bacillus firmus-I-4071. These results agree with that
substrates and in the course of this we identified potato
obtained by Kapoor et al., [23] who reported that, the
peels for cost-effective production of the enzyme under
members of the genus Bacillus and related genera are
study. SSF is receiving a renewed surge of interest,
known to produce extracellular pectinases, which have
primarily because increased productivity and prospect
applications in fiber industry. Bayoumi [6] produced
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J. Appl. Sci. Res., 4(12): 1708-1721, 2008
polygalacturonase from Bacillus cereus. Kobayashi et
conditions can stimulate the microbe to produce the
al. [27] purified the first bacterial Exo-PG from Bacillus
extracellular enzymes with different properties other
sp. strain KSM -P443. In the present study, Bacillus
than those of enzymes produced by the same organism
firmus-I-10104 was a Gram-positive rod, catalase
under the conditions performed in sub merged
positive, spore forming bacteria and grew in both
fermentation
[3 4 ]. In this field, many workers dealt with
aerobic and anaerobic conditions. These results are
the main different factors that affect the enzymes
connected with that recorded by Kapoor et al., [22] who
production such as temperature, pH, aeration, addition
found that, Bacillus sp. M G-CP-2 produce an alkaline
of different carbon and nitrogen sources. Although such
and thermostable PG in degumming of ramie
factors were previously studied by many authors, [29].
(Boehmeria nivea) and Sunn hemp (Crotalaria juncea).
Still, we need for more investigation seems to be
The environmental conditions in solid-state fermentation
continuously required to give a chance to isolate more
Fig 9: Relation between polygalacturonase productivity by Bacillus firm us-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.
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