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Ain Shams University Woman's the College Arts, Science &Education
IMPROVING CITRIC ACID PRODUCTION FROM SOME CARBOHYDRATES BY-PORODUCTS USING IRRADIATED
ASPERGILLUS NIGER
A thesisA thesisA thesisA thesis
Submitted for Ph.D Degree of science
in microbiology
to
the Botany Department
Women's College for Arts, Science & Education
Ain Shams University ,Cairo, Egypt
ByByByBy
Souzy Sobhy Farag B.Sc. of Science (Botany) Fac. of Sci, Ain Shames University 1982
M.Sc. of Sciences (Microbiology) Fac. of Sci, Zagazig University 2003
2011
Ain Shams University Woman's the College Arts, Science &Education
AcknowledgmentAcknowledgmentAcknowledgmentAcknowledgment
First, thanks for God for helping me to do this work.
I would like to express my deep gratitude to Prof. Dr. Fatma
Abd-El-Wahab Helimesh, professor of Microbiology, Women's
College, Ain Shams University Cairo, Egypt, for her kind supervising
this work and continuous valuable guidance.
My deep gratitude and sincerely thanks to Prof. Dr. Nahed Ali
Younis, Professor of Microbiology, Plant Research Department,
Nuclear Research Center, Atomic Energy Authority, for supervising
this work and her great efforts during the study, my sincers and cordial
appreciation.
Also I'm greatly indebted to thank with deep appreciation Dr.
Sherif Moussa Husseiny, Assistant Prof. of Microbiology Women's
College, Ain Shams University , Cairo, Egypt for suggesting the point
of the study, his kind supervision, and continuous guidance through
out the course of this work.
My sincere thanks are extended to Dr. Hany Wahib Botors,
Lecturer of Microbiology, Pant Research Department, Nuclear
research Center, for his cooperation during the study, My thanks to
Dr. Hanan Abd Alla Abdel Menem Lecturer of Biochemistry, Biological Application Department Nuclear Research Center for her
efforts in explanation PCR data also my thanks to all members of
Microbiology Unit for their useful help.
Finally, my sincere thanks are to the chairman of Nuclear Research
Center, and also to the Head of Plant Research Department – in
addition to Head of Plant and Microbiology Women's College, Ain
Shams University, Cairo, Egypt.
ABSTRACT
Twenty strains of A. niger were isolated from different sources,
screened for their capacity to produce citric acid. All the isolated strains were
able to produce citric acid in different quantities at different time intervals i.e.
4, 8 and 12 days on indicator medium. The best incubation period for
production for all isolates was 12 days. The most potent strains for production
were A1, A4 & A5, while A8, A16, A18 & A19 recorded weak production on that
medium. Citric acid productivity were obtained by all strains when using
different concentrations of four carbohydrate by-products (maize straw,
potato peel wastes, sugar beet pulp and molasses) when each used alone
without any additions after 12 days incubation and the production enhanced
when the fermentation medium amended with the same concentrations of the
mentioned substrates. Type and concentration of carbohydrate by-product
affect the production of citric acid by A. niger strains under the study.
Increasing substrate concentration led to increase in production, the best
concentration for production was 25% for all carbohydrate by-products. As
recorded with indicator medium, A1, A4 & A5 are also the most potent strains
for production when growing on the four carbohydrate by-products
supplemented to the basal medium, while A8, A6, A18 & A19 recorded the weak
production with the carbohydrate by-products used.
Production of the parental isolates A1, A4 & A5 on indicator medium
were: 0.96, 0.95 and 0.99 (mg/ml) respectively after 12 days incubation, while
maximum production by the obtaining resulting isolates (Treated by uv
irradiation) were: 1.78, 1.70 and 1.73 (mg/ml) from A4T2 (5min.), A4T1
(10min.) and A1T1 (5min.), respectively. Production of mentioned parental
isolates on basal medium with 25% maize straw were: 0.73, 0.70 and 0.85
respectively at the same incubation period, while maximum yield from the
obtained ones (Treated) were: 1.50, 1.45 (mg/ml) by A4T2 (5min.) and A4T1
(10min.), respectively. Production of parental selected isolates on basal
medium with 25% potato peel wastes: 0.94, 0.91 & 0.95 (mg/ml) respectively,
while maximum yield by the treated ones at the same medium were 1.85, 1.84
& 1.78 (mg/ml) by A1T1 (5min.), A1T2 (5min.) and A1T3 (5min.), respectively.
On basal medium with 25% sugar beet pulp parental production were 1.60,
1.50 and 1.51 (mg/ml), respectively after 12 days incubation, while maximum
yield by the derivatives at the same medium reached to 3.16 and 2.82 (mg/ml)
by: A1T1 (5min.) and A1T5 (min.). Parental isolates A1, A4 & A5 growing on
basal medium with 25% molasses recorded: 1.22, 1.04 and 1.20 (mg/ml),
respectively, while the yield reached from (5.00-5.62 mg/ml) for 17 obtained
treated ones from parental A1 after exposure time (2, 5, 10, 15 and 20 min.) to
uv irradiation with increases 4 folds approximately. Parental weak isolates
(A8, A16, A18 & A19) recorded 0.74, 0.70, 0.69 and 0.60 mg/ml, respectively on
indicator medium. Increase 8, 7, 6 & 5 folds by the treated ones. Maximum
production was 5.35 mg/ml recorded by A19T1 (10 min). Maximum production
obtained by γγγγ-rays in the study ranged from 2.00 – 2.40 mg/ml, increase 2 fold
on indicator medium and basal medium amended with the carbohydrate by-
products for some treated ones.
A1 & AT1 (5 min) also A19 & A19T1 (10 min) were chosen for PCR
study.RAPD-PCR analysis revealed that five positive unique markers
amplified by certain primers (mentioned in the study) identified the mutant
A1T1 (5 min), while in the wild isolate A1 there were nine unique markers
amplified by certain primers. Sixteen positive unique markers amplified and
identified the mutant A19T1 10 min, while in the wild isolate A19 twelve unique
markers were amplified by mentioned primers. The genetic similarity
between two genotypes A1 and its mutant A1T1 (5 min) is 81% and variation is
19% while the genetic similarity between A19 and its mutant A19T1 (10 min) is
73.6% and variation 26.4%.
In protein profile (SDS-PAGE) twenty two bands were obtained in A1
ranged from 243.2 to 30.1 KDa while its mutant A1T1 (10 min) have fifteen
bands ranged from 235.7 to 31.9 KDa. Also A. niger A19 showed twelve bands
ranged from 91.12 to 24.78 KDa while after uv irradiation 10 min mutant
obtained isolate A19T1 (10 min) showed sixteen bands ranged from 100.74 to
21.15 KDa.
CONTENTS Page
List of figures………...……………………………..…… --
List of tables ………..……………………………..…….. --
List of plates………..……………………………..……... --
ACKNOWLEDGEMENT
ABSTRACT …….………………………………………. I
INTRODUCTION………………………………………. 1
AIM OF THE WORK …………………………………... 4
REVIEW OF LITERATURE …………………………… 5
Natural citric acid ……………………………………… 5
Citric acid cycle …………………………………………. 5
Manufacture citric acid ....................................................... 6
Industrial importance of citric acid ..................................... 6
Biosynthesis of citric acid ................................................... 8
The possibility of utilizing carbohydrate by-products for
citric acid production by A. niger ................................... 11
Isolation of mutants of A. niger for hyperproduction of citric
acid through uv light irradiation……….....................…….. 14
Isolation of mutants of A. niger for hyperproduction of citric
acid through γ rays ..................................................... 17
MATERIALS AND METHODS .................................... 21
Microorganisms (parental strains) ...................................... 21
Inoculum preparation ……………………………………. 21
Carbohydrate by-products ................................................. 22
Cultivation .......................................................................... 22
Exposure of selected parental strains of Aspergillus niger
to uv light irradiation and γ-rays: .................................... 22
Isolation of obtained strains (treated isolates)..................... 23
Assay of citric acid ............................................................ 23
Random Amplified Polymorphic DNA Polymerase Chain
Reaction (RAPD-PCR) ....................................................... 24
Sodium dodecyl sulfate (SDS) - Polyaciylamide gel.......... 27
Protein electrophoresis........................................................ 31
RESULTS …………………………………….…………. 35
Part I: Selection the most potent strains of A. niger growing on
a modified Czapek's agar medium supplemented by
different carbohydrate by-products for citric acid
production………………………………………. 34
1. Production of citric acid by Aspergillus niger strains on
indicator medium............................................................. 35
2. Production of citric acid by Aspergillus niger strains on
maize straw after 12 days incubation.............................. 40
3. Production of citric acid by Aspergillus niger isolates on
potato solid wastes (peels) after 12 days incubation........ 44
4. Production of citric acid by Aspergillus niger isolates on
sugar beet pulp after 12 days incubation........................... 44
5. Production of citric acid by Aspergillus niger isolates on
molasses after 12 days incubation..................................... 48
Part II: Yield of citric acid by treated (potent & weak)
strains of Aspergillus niger that growing on
different carbohydrate by-products using uv light
irradiation & γ-rays ............................................... .. 62
1. Influence of uv light irradiation & γ -rays on the most
potent strains A. niger for citric acid production............. 63
A. U.V light irradiation..................................................... 63
B. γ-rays ........................................................................... 75
2. Influence of uv light irradiation on weak isolates of
A. niger of citric acid production..................................... 91
Part III: RAPD-PCR amplification and protein
electrophoresis for A. niger isolates.......................100
DISCUSSION ……………………………………………. 126
SUMMARY……………………………………………… 136
CONCLUSION …….……………………………………. 144
RECOMMENDATION ………………………………... 145
REFERENCES …………………………………………. 146
ARABIC SUMMARY…………………………………….
List of Figures
Fig. Subject Page
1 Standard curve citric acid. 24
2 Production of citric acid (mg/ml) by different
strains of Aspergillus niger using indicator
medium after 12 days incubation
38
3 Maximum Citric acid production by A. niger
isolates (A1, A4 & A5) on indicator medium at
different time intervals
39
4 Weakest production of citric acid by four isolates
(A8, A16, A18 & A19) on indicator medium at
different time intervals
39
5 Citric acid production (mg/ml) by different
isolates of A. niger growing on 25% maize straw
after 12 days incubation
43
6 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal medium
amended with 25% maize straw after 12 days
incubation
43
7 Citric acid production (mg/ml) by different
isolates of A. niger growing on 25% of potato
solid wastes after 12 days incubation
47
8 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal medium
amended with 25% of potato solid wastes after 12
days incubation
47
9 Citric acid production (mg/ml) by different
isolates of A. niger growing on 25% of sugar beet
pulp after 12 days incubation
51
10 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal liquid
medium amended with 25% of sugar beet pulp
after 12 days incubation
51
11 Citric acid production (mg/ml) by different
isolates of A. niger growing on 25% of molasses
after 12 days incubation
54
List of Figures (Cont.)
Fig. Subject Page
12 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal liquid
medium amended with 25% of molasses after
12 days incubation
54
13 Citric acid production (mg/ml) by most potent
isolates on basal medium amended with
different concentration of maize straw
56
14 Citric acid production (mg/ml) by weak
isolates on basal medium amended with
different concentration of maize straw
56
15 Citric acid production (mg/ml) by most potent
isolates on basal medium amended with
different concentration of potato solid wastes
57
16 Citric acid production (mg/ml) by weak
isolates on basal medium amended with
different concentration of potato solid wastes
57
17 Citric acid production (mg/ml) by most potent
isolates on basal medium amended with
different concentration of sugar beet pulp
58
18 Citric acid production (mg/ml) by weak
isolates on basal medium amended with
different concentration of sugar beet pulp
58
19 Citric acid production (mg/ml) by most potent
isolates on basal medium amended with
different concentration of molasses
59
20 Citric acid production (mg/ml) by weak
isolates on basal medium amended with
different concentration of molasses
59
List of Figures (Cont.)
Fig. Subject Page
21 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after uv irradiation after 12 days incubation on
indicator medium
68
22 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after uv irradiation after 12 days incubation on
indicator medium
68
23 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after uv irradiation after 12 days incubation on
indicator medium
68
24 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% maize straw
70
25 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% maize straw
70
26 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% maize straw
70
List of Figures (Cont.)
Fig. Subject Page
27 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% potato solid
wastes
72
28 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% potato solid
wastes
72
29 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% potato solid
wastes
72
30 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% sugar beet
pulp
74
31 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% sugar beet
pulp
74
32 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% sugar beet
pulp.
74
List of Figures (Cont.)
Fig. Subject Page
33 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
77
34 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
77
35 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
77
36 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on indicator liquid medium
81
37 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after gamma rays irradiation rays after 12 days
incubation on indicator liquid medium
81
38 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on indicator liquid medium
81
39 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% maize straw
83
List of Figures (Cont.)
Fig. Subject Page
40 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% maize straw
83
41 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% maize straw
83
42 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% potato solid wastes
86
43 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% potato solid wastes
86
44 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% potato solid wastes
86
45 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% sugar beet pulp
88
List of Figures (Cont.) Fig. Subject Page
46 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% sugar beet pulp
88
47 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% sugar beet pulp
88
48 Citric acid production (mg/ml) of selected
parental isolate A1 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% molasses
90
49 Citric acid production (mg/ml) of selected
parental isolate A4 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% molasses
90
50 Citric acid production (mg/ml) of selected
parental isolate A5 and its obtained isolates
after gamma rays irradiation after 12 days
incubation on basal medium amended with
25% molasses
90
51 Citric acid production (mg/ml) of parental
weak isolate A8 and its obtained isolates after
uv irradiation after 12 days incubation on
indicator medium
96
52 Citric acid production (mg/ml) of parental
weak isolate A16 and its obtained isolates after
uv irradiation after 12 days incubation on
indicator medium
96
List of Figures (Cont.) Fig. Subject Page
53 Citric acid production (mg/ml) of parental
weak isolate A18 and its obtained isolates after
uv irradiation after 12 days incubation on
indicator medium
96
54 Citric acid production (mg/ml) of parental
weak isolate A19 and its obtained isolates after
uv irradiation after 12 days incubation on
indicator medium
97
55 Citric acid production (mg/ml) by A19 (T1 uv
10) of A. niger on different carbohydrate by
products after 12 days incubation.
97
56 Substrate consumed (%) by A19 (T1 uv 10) of
A. niger on different carbohydrate by products
after 12 days incubation
97
57 RAPD banding patterns amplified for A. niger
(A1) and its obtained isolate(A1T15min) using
primers(OPO-14&OPO-02) M (DNA marker)
= 5Kb ladder
104
58 RAPD banding patterns amplified for A. niger
(A1) and its obtained isolate (A1T15 min) using
primers(OPB-10&OPB-08) M (DNA marker)
= 1Kb ladder
104
59 RAPD banding patterns amplified for A. niger
(A1) and its obtained isolate (A1 T1 5min)
using primer (OPA-05)M (DNA marker) =
1Kb ladder.
105
60 RAPD banding patterns amplified for A. niger
(A1) and its obtained isolate (A1T1 5min) using
primers (OPA-04, OPA-15 & OPA-18) M
(DNA marker) = 1Kb ladder
105
List of Figures (Cont.)
Fig. Subject Page
61 RAPD banding patterns amplified for A. niger
(A1) & its obtained isolate (A1 T1 5min) using
primers (OPC-14 &OPB-05)M (DNA marker)
= 1kb ladder.
106
62 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10 min)
using primer OPG-0.5 M (DNA marker) = 1kb
ladder
108
63 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10min)
using primer OPH-15 M (DNA marker) = 100
base pairs ladder.
109
64 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10min)
using primer OPC-10 M (DNA marker) = 1kb
ladder.
109
65 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolated (A19 T1 10min)
using primer OPC-20 M (DNA marker) = 1
kb ladder
110
66 RAPD banding patterns amplified for A niger
(A19) and its obtained isolate (A19 T1 10 min)
using primer OPA-20 M (DNA marker) = 1kb
110
67 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10min)
using primer OPO-04 M (DNA marker) = 1Kb
111
68 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10min)
using primer OPB-15 M (DNA marker) = 1kb
ladder.
111
List of Figures (Cont.)
Fig. Subject Page
69 RAPA banding patters amplifying for A. niger
(A19) and its isolate (A19 T1 10min) using
primer OPB-05 M (DNA marker) = 1kb
ladders
112
70 RAPD banding patterns amplified for A. niger
(A19) & obtained isolate (A19 T1 10min) using
primer OPB-07 M (DNA marker) = 100 base
ladder
112
71 RAPD banding patterns amplified for A. niger
(A19) and its obtained isolate (A19 T1 10min)
using OPB-06 M (DNA marker) = 1kb ladder
113
72 Marker Lane Profile 121
73 Lane A1 T1 Profile 122
74 Lane A1 Profile 123
75 Lane A19 T1 Profile 124
76 Lane A19 Profile 125
List of Tables
Table Subject Page
1 Source of isolation for twenty strains of A.
niger
36
2 Diameter of clear zone (mm) and production
of citric acid (mg/ml) by different strains of
Aspergillus niger using indicator medium at
different time intervals
37
3 Citric acid production (mg/ml) by different
isolates of A. niger grown on different
concentrations of maize straw after 12 days
incubation
41
4 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal medium
amended with different concentrations of
maize straw after 12 days incubation
42
5 Citric acid production (mg/ml) by different
isolates of A. niger grown on different
concentrations of potato solid wastes after 12
days incubation
45
6 Citric acid production (mg/ml) by different
isolates of A. niger grown on basal medium
amended with different concentrations of
potato solid wastes after 12 days incubation
46
7 Citric acid production (mg/ml) by different
isolates of A. niger growing on different
concentrations of sugar beet pulp after 12 days
incubation
49
8 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal liquid
medium amended with different concentra-
tions of sugar beet pulp after 12 days
incubation
50
List of Tables (Cont.)
Table Subject Page
9 Citric acid production (mg/ml) by different
isolates of A. niger growing on different
concentrations of molasses after 12 days
incubation
52
10 Citric acid production (mg/ml) by different
isolates of A. niger growing on basal liquid
medium amended with different concentra-
tions of molasses after 12 days incubation
53
11 Number of colonyforming units of selected
isolates of A. niger (A1, A4, & A5) on indicator
medium after exposure to uv irradiation
64
12 Screening of treated isolates obtained after uv
irradiation of selected A. niger isolates (A1, A4
and A5) for production of citric acid on
indicator medium in terms of diameters of
clearing zone (mm) at different time intervals
65
13 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after uv
irradiation on indicator medium
67
14 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after uv
irradiation growing on basal liquid medium
amended with 25% maize straw
69
List of Tables (Cont.)
Table Subject Page
15 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after uv
irradiation growing on basal liquid medium
amended with 25% potato solid wastes
71
16 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after uv
irradiation growing on basal liquid medium
amended with 25% sugar beet pulp
73
17 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after uv
irradiation growing on basal liquid medium
amended with 25% molasses
76
18 Number of colony-forming units of selected
isolates of A. niger (A1, A4 and A5) on
indicator medium after exposure to gamma
rays
78
19 Screening of treated isolates obtained after
gamma rays irradiation of selected A. niger
isolates (A1, A4 and A5) for production of
citric acid and obtained isolates on indicator
medium in terms of diameter of clearing zone
(mm) at different time intervals
79
20 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after gamma
rays irradiation on indicator liquid medium
80
List of Tables (Cont.)
Table Subject Page
21 Citric acids production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after gamma
rays irradiation growing on basal liquid
medium amended with 25% maize straw
82
22 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after gamma
rays irradiation growing on basal liquid
medium amended with 25% potato solid
wastes
85
23 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after gamma
rays irradiation growing on basal liquid
medium amended with 25% sugar beet pulp
87
24 Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5)
and obtained isolates (treated) after gamma
rays irradiation growing on basal liquid
medium amended with 25% molasses
89
25 Number of colony-forming units of weak
isolates of A. niger (I8, I16, I18 and I19) on
indicator medium after uv irradiation
93
26 Screening of treated isolates obtained after uv
irradiation of parental weak isolates of A.
niger (A8, A16, A18 and A19) for production of
citric acid on indicator medium in terms of
diameter of clearing zone (mm) at different
time intervals
94
List of Tables (Cont.)
Table Subject Page
27 Citric acid production by parental weak
isolates (A8, A16, A18 and A19) of A. niger and
obtained isolates after uv irradionlion on
indicator medium at different time intervals
95
28 Citric acid production (mg/ml) by A19 (T1 uv
10) of A. niger on different carbohydrate by
products after 12 days incubation
98
29 Number of amplified DNA bands scored for
the wild strain A. niger A1 (potent isolate) and
its obtained isolate (A1 T1 5min) by uv
irradiation.
102
30 Positive and negative unique RAPD markers
and their molecular weight of the two strains
of A. niger (A1 & A1 T1 5 min).
103
31 Number of amplified DNA bands scored for
the wild strain of A. niger A19 and its obtained
isolate (A19T1 10 min) by uv irradiation.
107
32 Positive and negative unique RAPD markers
and their molecular weight for wild A. niger
A19 (weak isolate) and its obtained isolate
(A19T1 10min) after treatment with uv
irradiation.
115
33 RAPD based genetic similarity (GS) between
genotypes of A. niger: (A1 & A1 T1 5 min)-
(A19 & A19T1 10min).
116
34 SDS-PAGE for (A1 &A1 T1 5 min and A19 &
A19 T1 10 min.)
118
List of Plates
Plates Subject Page
1,2 & 3 Most potent isolates of A. niger for citric
acid production on indicator medium after
12 days incubation
60
4, 5, 6 & 7 Weak isolates of A. niger for citric acid
production on indicator medium after 12
days incubation
61
7 Wild (Parental isolate) of A. niger (I 19) on
indicator medium after 12 days incubation
99
8 Derivative isolate of A. niger (I 19) after
exposure to uv light irradiation (10 minutes)
after 6 days incubation on indicator medium
(A19 T1).
99
- 1 -
Introduction
Citric acid (C6 H5 07) i.e., 3 hydroxy 1, 3, 5 propane
tricarboxylic acid is ubiquitous in nature and exists as an
intermediate in citric acid cycle when carbohydrates are oxidized
to carbon dioxide. It is solid at room temperature, melts at 153°C
and decomposes at higher temperature into other products
(Rojoka et al., 1998). It is responsible for tart taste of various
fruits in which occurs i.e. lemon, lime, figs, oranges, pineapples,
pears and gooseberries (Francis, 2000). It is non toxic and easily
oxidized in the human body. Because of its high solubility,
palatability and low toxicity, it can be used in food, biochemical
and pharmaceutical industries, these uses have placed greater
stress on increased citric acid production and search for more
efficient fermentation process. The world wide demand of citric
acid about 6.0 x105 tons per year and is bound to increases day by
day (Ali et al., 2001).
In 2004, the world wide production of citric acid was
approximately 1.4 million tons according to the business
communications Co. (BCC)'s recent studies of fermentation
(Soccol et al., 2006). Moreover due to its large application and
low price, the citric acid consumption is expected to grow
significantly until 2009, and this raises the need for industries to
search for new technological alternative and for cost reduction in
citric acid production (Vandenberghe et al.,2004) citric acid
(CA) has a variety of applications, 70% of it used in food and
- 2 -
beverage industries, 12% in the pharmaceutical industry and 18%
in other industries (Soccol et al., 2003 Pandey et al., 2001). A
potentially promising economical and efficient source for
producing citric acid is to use the fungus A. niger due to its
higher capacity to accumulate acid when compared to other
organisms (Adham, 2002; Pazauki et al., 2000, Pera and
Callieri, 1999; Maddox and Brooks, 1998 and Fiedurk et al.,
1996 and Yokoya, 1992). It has been found to be an efficient
producer of citric acid when cultured media comprised of residues
and extracts such as sugar and molasses (Mehyar et al.,2005;
Bayraktar and Mehmetaglu, 2000; El-Holi, 1999; Roukas,
1998; 1991; Gutierrez Rojas, 1995; Gupta and Sharma, 1994
and Khare et al.,1994).
Possibilities have been done for using some by-products
and agroindustrial residues in citric acid production by solid state
fermentation, on the other hand, solving in serious environmental
problems (Rodrigues et al., 2010; Darani and Zoghi, 2008;
Soccol and Vandenberghe, 2003; Prado, 2002; Pintado et
al.,1998; Soccol, 1996, and Kolicheski 1995). Citric acid
production by A. niger depends on several specific nutritional and
environmental conditions as well as the particular strain of
microorganisms. It is a primary metabolite of Kreb's cycle
production from reacting pyruvate with acetyl CoA which is
catalyzed by pyruvate carboxylase (Hagedorn and Kaphammer
1994; Stanburry and Whitaker 1984).
- 3 -
Mutations are abrupt and so are the hereditary
modifications in the genetic material. The organism containing
the DNA are not static molecules and their bases are frequently
exposed to natural or artificial agents can cause modification in
their structure or in chemical composition (Griffiths et al., 2006
and Zaha 2003). The increase in citric acid productivity has been
achieved using mutation and strain selection. Mutation strains
with certain characteristics such as enhanced citric acid
production and increased fermentation rate have been previously
selected after submitting the genetic material to physical or
chemical mutagenic agents (Lotfy et al., 2007, Griffiths et
al.,2006, Ikram-Ul Hag et al.,2001 and Rohr et al.,1983).
The most frequently used method is induction by UV
irradiation that can randomly provide a strain with a higher
capacity of citric acid production when compared to the control
strain (Griffiths et al.,2006 and Zaha, 2003). γ-rays as
mutagenic agent used for improve citric production by A. niger
(Alani et al.,2007) investigated optimization of citric acid
production from a new strain and mutant of A. niger after γ-ray
irradiation using solid state fermentation. In addition Parvez et
al.,1998 obtained mutant strain of A. niger for citric acid
production after γ-irradiation.
- 4 -
- 5 -
Aim of the work
Accordingly, this work was undertaken to through light upon
the following aspects:
1. Isolation and screening of different strains of A.niger for their
ability to grow and produce citric acid.
2. Selection of the most potent and weak isolates for citric acid
production.
3. Utilization of different carbohydrate by-products as substrates
for citric acid production by isolates of A.niger.
4. Studying of the influence of mutagenic agents (uv light
irradiation and γ-rays) on the most potent and weak isolates.
5. Quantitative estimation of citric acid production by irradiated
strains that allows to grow on carbohydrate by-products as
carbon and energy source.
6. Studying of some molecular studies on irradiated strains
- RAPD-PCR
- protein profile.
- 6 -
- 7 -
Review of Literature
Natural citric acid:
Bouchard & Merritt, 1979 reported that the total
circulating citric acid in the serum of man is approximately
1mg/kg of body weight. Beli & Odian, 2000 and Francis, 2000
showed that citric acid is found naturally in almost all living
things both plant and animal. It is predominant acid in substantial
quantity' in citrus fruits (oranges, lemons, limes, etc..,), in berries
(strawberries, raspberries) and in pineapples. Citric acid is also
predominat acid in many vegetables such as potatoes, tomatoes,
asparagus, turnips and peas but in lower concentrations. Nill,
2002 observed that some plants naturally release citric acid from
their roots into the surrounding soil, in order to "bind" aluminum
ions that are present in some soils, such aluminum which slows
plant growth and decrease crop yields is present to a certain
degree (which causes at least some crop yield reduction). CH2 COOH |
HO C – COOH |
CH2 COOH
Citric acid (3-hydroxyl 1, 3, 5 propane-tricarboxylic acid)
Citric acid cycle:
A system of enzymatic reactions in which acetyl residues
are oxidized to carbon dioxide and hydrogen atoms. It is also
- 8 -
known as the tricarboxylic acid cycle (TCA cycle) or Kerbs cycle
occurs in almost all aerobic (air requiring) organisms (Nill, 2002).
Manufacture of citric acid:
Francis, 2000 reported that citric acid is manufactured by
fermentation, a natural process using living organisms. The acid is
recovered in pure crystalline form either as the anhydrous or
monohydrate crystal depending on the temperature or
crystallization. The transition temperature is 36.6°C.
Crystallization above this temperature produces an anhydrous
product while the monohydrate forms at lower temperatures. The
anhydrous form is preferred for its physical stability and its more
widely available commercial form.
Industrial importance of citric acid:
Higgins et al., 1985 reported that citric acid has a pleasant
acid taste and is soluble in water, it is widely used in food
industry, in pharmaceuticals and cosmetics, the esters of the acid
are used in the plastic industry and as a metal chelator, citric acid
finds application in metal purification and as a readily
biodegradable ingredient of detergents. Kishore & Smis, 1993;
Pallares et al., 1995 mentioned that citric acid is widely used in
the food, pharmaceutical and chemical industries due to its
distinctive properties as an acidulent, flavoring agent, antioxidant
and high solubility.
- 9 -
Francis, 2000 mentioned to the broad spectrum for using
citric acid and its salts in food and beverage products. i.e.,
beverages, gelation desserts, backed goods, jellies, jams,
preserves, candies, fruits and vegetables, dairy products, meats,
seafood and fats & oils. Soccol et al., 2003 pointed that buffering
properties of citrates are used to control pH in household cleaners
and in pharmaceuticals. Luciana et al., 2004. showed that the
food industry is the consumer of citric acid using almost 70% of
the total production followed by about 12% by the pharmaceutical
industry and 18% for other applications.
Xie & West, 2006 said that the estimated world production
of citric acid was reported as 1.000.000 tons/year and the world
market demand is increasing day by day. Lotfy et al., 2007
concluded that citric as is one of the of the few bulk chemicals
produced by fermentation. It has a broad use in the household, in
the preparation of numerous industrial products and in many
industrial areas such as the food, pharmaceutical, and chemical
industries, and as a cleaning agent.
Biosynthesis of citric acid:
Different microorganisms had the ability to synthesize
citric acid. Among these microorganisms that considered as active
producers of citric acid: Aspergillus niger, penicillium lactum,
Mucor pyri formis, Tricoderma viride, (Klrimura et al., 1990).
- 10 -
The variety of yeasts known to produce citric acid from
various sources are species of Candida, Saccharomyces,
Yarrowia, (Good et al., 1985; El-Sayed 1986; Kishore & Smis,
1993 and Crolla & Kennedy, 2001). Arzumanov et al., 2000
reported that various strains of fungi which have been found to
accumulate citric acid in their culture media include strains of A.
niger, A. awamori, Penicillium restrictum, Trichloderma viride,
Mucor Piriformis and Yarrowia Lipolytica, but A. niger remained
the organism of choice for the production of citric acid.
Mourya and Jauhri, 2000 demonstrated that A. niger has
been the organism of choice due to its ease of handling, ability to
use a variety of cheap raw materials and high yield of citric acid,
also they screened seventeen strains of A. niger for their capacity
to produce citric acid using starch hydrolysate as a substrate, the
most efficient strain TICC-605 was selected for further
improvement in citric acid content by
- 11 -
The citric acid cycle. Two carbon atoms enter the cycle in the form of acetyl-CoA and are joined to oxaoacetate to form citrate (Step 1). During the series of reaction that then occur, two molecules of carbon dioxide are formed (Steps 5 and 6), and four pairs to of hydrogens are produced and carried to the respiratory chain (Steps 4, 6, 8 and 10). At the end to the cycle a molecule of oxaoacetate is formed, ready for another turn of the cycle (Step 10).
- 12 -
mutation. Hang and Woodams, 2001 studied enzymatic
enhancement of citric acid production by A. niger from corncobs.
Ishaq et al., 2002 studied time course profile of citric acid
fermentation by A. niger and its kinetic relations and found that
mutant strain of A. niger GCB47 was a faster growing organism
and has the ability to hyper produce citric acid. Asad et al., 2003
evaluated phosphate limitation for enhanced citric acid
fermentation using A. niger mutant uv- µ9 on semi-pilot scale.
Kurbanoglu, 2004 studied the potential use of ram horn
hydrolysate (RHH) as a supplement for improvement of citric
acid production by A. niger NRRL 330. Prado, et al., 2005
worked to study the relation between citric acid production and
respiration of A. niger LPB 21 in solid state fermentation of
cassava baggasse, the experiment was carried out in horizontal
drum bioreactor coupled with gas chromatography system. Kim
et al., 2006 studied nutrient optimization for the production of
citric acid by A. niger NRRL 567 grown on peat moss enriched
with glucose.
Lotfy et al., 2007 demonstrated to citric acid production by
a noval A. niger isolate and studied the optimization of process
parameters through statistical experimental designs. Barrington
and Woo, 2008 found that maximum citric acid production in
- 13 -
optimized condition by central composite design (CCD)
represented about a 2.7 fold increase compared to that obtained
from control before optimization when A. niger NRRL 567 grown
in peat moss. Rodrigues et al., 2010 aimed to optimize the
physical-chemical conditions of citric acid through a careful strain
selection of A. niger by solid state fermentation, the best
production was reached at 65%. moisture, 30oC and pH 5.5.
The possibility of utilizing carbohydrate by-products for citric
acid production by A. niger:
Considerable interest has been developed in using
agricultural wastes as substrates for citric acid production
including pineapple wastes (Tran & Mitchell 1995 and Tran et
al., 1998), grape pomace (Hang & Woodams 1995), apple
pomace (Fatemi & Shojaosadati, 1999; Shojaosadati &
Babaripour, 2002), corn cobs (Hang and Woodams 2001), kiwi
fruit peel (Kumar et al., 2003), cassava bagasse (Vandenberghe
et al., 2004 & Flavera et al., 2005).
Xie & West, 2006 screened seven citric acid producing A.
niger for their ability to synthesize citric acid on untreated and
autoclaved wet corn distillers grains by solid-state fermentation.
The most effective citric acid producing strain of A. niger was
ATCC 9142 on the untreated or autoclaved grains. The
- 14 -
autoclaved grains supported less citric acid production by the
majority of strains screened.
Different agro-industrial residues have been investigated as a
substrate for citric acid production. El-Batal et al., 1995 studied
five strains of Aspergillus niger (EMCC 102, EMCC 104, EMCC
111, EMCC 132 and EMCC 147) were used for citric acid
production at different incubation periods using different cheap
carbohydrate substrates, such as beet, cane, citrus molasses and
milk whey. A. niger EMCC111 was found to be the most potent
strain for citric acid production from beet molasses after 11 days
of incubation at 30oC.
Ikram-ul-Hag et al., 2003, studied production of citric
acid from raw starch by Aspergillus niger. Shake flask and semi-
solid culture methods were compared using A. niger GCB-47
(parental strain) and GCMC-7 (mutant strain). In shaking culture
with 150g/L soluble starch as a carbon source, the mutant strain
GCMC-7 produced 69.5 g/L citric acid, which was 1.48 fold
greater than the parental strain GCB-47. Direct production of
citric acid from corn and potato starch was examined using semi-
solid culture. It was observed that the mutant strain was a faster
growing organism. The mutant strain GCMC-7 produced 71.4 and
92.9 g/L citric acid approximately 1.12 and 1.44 times as much as
the parental strain GCB-47 from 200 g/L corn and potato starch,
- 15 -
respectively. The finding suggest that GCMC-7 possesses
enhanced ability for sugar metabolism and citric acid production.
Asad et al., 2003 evaluated twenty five strains of A. niger,
isolated from different soil samples for citric acid production
using cane-molasses medium. Vandenberghe et al., 2004
evaluated three different agroindustrial wastes, sugar cane
bagasse, coffee husk and cassava bagasse for their efficiency in
production of citric acid by a culture of Aspergillus niger under
solid-state fermentation. Cassava bagasse supported best fungal
growth giving the highest yield of citric acid among the tested
substrates. Results showed that the fungal strain had good
adaptation to the cassava bagasse substrate and increased the
protein content in the fermented matter.
Lotfy et al., 2007 studied reduction of the fermentation
medium cost, corn steep liquor and calcium phosphate pre-treated
beet molasses were successfully used as a subsituents of nitrogen
and carbon sources in the growth medium of A.niger UMIP 2564
respectively. These medium substitutions resulted in citric acid
fermentation culture with a product yield of 74.56%.
Isolation of mutants of A. niger for hyperproduction of citric
acid through uv light irradiation:
Hamissa et al., 1992 used different doses of uv irradiation
to obtain mutants of Aspergillus niger 20 for raising citric acid
- 16 -
(CA) production. The uv treatment resulted in the development of
31 isolates, some of which differed in their morphology and
sporulation ability. Irradiation for 12 min. did not improve CA
yield by the developed mutants. Nine mutants gave significantly
higher yields of CA than parent culture when exposed to uv
treatment for 18 min. (6 mutants) and 24 min. (3 mutants). The
highest yield of CA obtained after exposure 18 min. (26.08%
higher than the parent culture) was that produced by isolate 5 uv
18. Irradiation for 24min. appeared to be the best treatment for
inducing mutation with isolate 19 uv 24 which produced a
32.13% higher CA yield than the parent control. When these nine
active mutants were tested for their CA productivity in the
presence of potassium ferrocyanide, only the isolates 5 uv 18 and
21 uv 24 each yielded 3% more CA than the original culture on a
sugar basis.
Rugsaseel et al., 1993 induced mutants with enhanced
citric acid production from soluble starch from Aspergillus niger
wu-2223L. after uv irradiation of a conidia suspension of strain
wu-2223L, mutants were selected on modified starch-methyl red
agar plates on the basis of higher amylolytic activity and acid
productivity. The 8 mutants selected showed enhanced citric acid
production from soluble starch in shaking culture. Among them, a
representative mutant strain, 2M-43, produced 48.0 g/L of citric
acid from 120 g/L of soluble starch in qd of cultivation in shaking
- 17 -
culture, whereas strain wu-2223L produced 35.1 g/L.
Glucoamylase activities in the culture filtrates of strains 2M-43
and wu-2223L reached maximum level of 3.62 u/ml and 2-11
u/ml, respectively, both at 3d of cultivation, and thereafter
decreased.
Sarangbin and Watanapokasin, 1999 carried out
selection of protease-negative mutant strains of A. niger in semi-
solid culture in order to enhance citric acid production from yam
bean. The protease-negative mutants were obtained by uv
irradiation of the parental strain Yang no. 2 using a halo selection
medium, a number of mutants with decreased extra cellular
protease activity were selected. Citric acid productivity by the
selected mutant strains was tested on a modified starch-methyl red
agar plate. The best mutant strain Yw-122 was obtained and
produced 106 g/L of citric acid, whereas the parental strain Yang
no 2 produced 58 g/L, from 140 g/L of soluble starch in semi-
solid culture at 5 days of cultivation time. During the whole
period of cultivation when 17 g/ plate of red-shaped yam bean
was used instead of soluble starch, the protease-negative mutant
strain Yw-112 produced 490 g/L of citric acid, which is
approximately 1.5 times as much citric acid as Yang no 2
produced.
- 18 -
Conte & Marine, 2003 induced mutants of Aspergillus
niger N402, by uv mutagenesis, were selected and tested for
resistance or sensitivity to 5-fluorocytosine. Some mutants
showed increased citric acid production, which did not correlate
with the intracellular amount of protein or ammonium ion. The
resistance to 5-fluorocytosine proved to be a rational approach for
isolation of new mutants with improved production of citric acid.
The best mutant (FR13) accumulated double the amount of citric
acid produced by the parental strain. Pewlong et al., 2003 used
ultraviolet and gamma irradiation to induce mutation of A. niger
ATCC11414 to increase ability of citric acid production. Five
mutants of high-producing citric acid were 7 uv-18, A2-14, 9 uv-2,
9 uv 27 and 9 uv-10. The yield of citric acid was 2.0 to 3.84 fold
higher than that of the wild type strain.
Goulart and Marin, 2005 studied some filamentous fungi
present the phenomenon of dimorphism, their morphological
structure alterations being capable of including metabolism
changes. The A. niger strain 10v10, a producer of citric acid, was
submitted to the mutagenic action of ultraviolet irradiation which
respectively selects mutants sensitive or resistant to the antifungal
agent 5-fluorcytosine (5-FC). 5-FC sensitive mutants presented a
morphological alteration to a yeast-like form. Morphological
reversal to the filamentous form was observed only. The presence
- 19 -
of Ca Cl2 (500 mµ) for the mutants strains/ and 2 while the acid
production occurred in both, yeast-like and filamentous forms.
Rodrigues et al., 2010 obtained eleven mutant strains with
uv irradiation of A. niger LPBBC, tested in SSF where two
mutants showed a higher citric acid (CA) production when
compared to the parental strain, A. niger LPBB produced 537.6 g
of citric acid / kg of citric pulp (CP) on the sixth day of
fermentation, while A. niger LPBB6 produced 616.5 g of CA/ kg
of CP on fourth day of fermentation representing a 19.5%. and
37% gain, CA production has been conducted through a careful
strain selection, physical-chemical optimiza-tion and mutation.
Isolation of mutants of A. niger for hyperproduction of citric
acid through γγγγ rays:
Das & Nandi, 1972 treated Aspergillus niger with uv and
gamma irradiation (60Co). Mutagenic treatments resulted in
substrains with either increased or decreased citric acid
production, while some were unchanged in this respect. Low-
yielding strains predominant in all the experiments. Analysis of
the superior substrains showed that gamma irradiation was most
effective in producing the greatest number of superior mutants.
Islam et al., 1984 investigated semi-pilot scale production
of citric acid with a-gamma-ray induced mutant (HB3) of
Aspergillus niger using 500, 1000 and 1500ml medium in 51
- 20 -
fermentation jars. Yield of citric acid was found to be seven-fold
higher compared to the parent in 1000ml medium and the
corresponding increase was two-fold in the 500ml medium. With
1500 ml/ formation jar the yield was low with both the parent and
the mutant strain, through the mutant gave higher yield compared
to the parent. Islam, 1990 subjected conidia of the citric acid
fermenting fungal mutant Aspergillus niger 14/20 to the gamma
radiation treatment. Mutants giving higher total triable acid values
than their parent strain were selected and tested under varied
environmental and cultural conditions. After screening of about
2000 mutants, two of them with the highest yield of citric acid in
molasses medium (60-76 gm/ml) were selected for further
experiments.
Begum et al., 1990 induced mutants of A. niger by γ-rays
in different carbohydrate media, they isolated a natural strain of
A. niger CA16, and two of its second step mutants, 136/40 and
277/30, grown on different sugar substrates gave maximum citric
acid yields of 34, 70, and 126 mg/ml respectively in sucrose
medium. Combination of two sugars in the medium at 50% of
each improved the yields of citric acid for the sucrose: glucose,
glucose: sorbitol, glucose: xylose and xylose: sorbitol
- 21 -
combinations with the mutant strains. Inclusion of galactose in
combinations decreased the citric acid yield.
Begum et al., 1991 studied effect of cultural pH and
incubation temperature on citric acid yield and kinetic patterns of
citric acid fermentation by a natural isolate of A. niger as CA 16
and one of its gamma ray induced mutants using cane molasses as
growth and fermentation substrate. Mutant strain, 277/30 gave
maximum citric acid yield of 35 g/L at pH 3.5 and 28oC in
molasses medium. Parent strain, CA 16 gave a maximum yield of
34 g/L at pH 4.0 and 26oC in molasses medium adjusted to 16%
sugar and 100% Prescott salt in the medium. In kinetic studies,
strains showed combinations kinetics of citric acid fermentation
where product formation is directly related to growth and cell
mass and indirectly related to carbohydrate uptake.
El-Batal et al., 1995 exposed inocula of A. niger
EMCC111 to doses (0.05 – 0.8 KGy) of γ-ray and showed that the
dose 0.4 kGy was the optimum for maximum citric acid
production. Parvez et al., 1998 studied citric acid production
from sugar cane molasses by A. niger NTAB 280 in a batch
cultivation process. A maximum of 90 g/L total sugar was utilized
in citric acid production medium. From the parental strain A.
niger, mutant strains, showing resistance to 2-deoxyglucos in
- 22 -
vogal's medium containing molasses as a carbon source were
induced by γ-irradiation. Among the new series of mutant strains,
strain RP7 produced 120 g/L while the parental strain produced
80 g/L citric acid (1.5 fold improvement) from 150 g/L of
molasses sugars. The period of citric acid production was
shortened from 10 d for the mild-type strain to 6-7 d for the
mutant strain. Also the mutant grew faster than its parent. This
indicated that the selected mutant is insensitive to catabolite
repression by higher concentrations of sugars for citric acid
production.
- 23 -
Materials and Methods
Microorganisms (parental strains):
Twenty strains of Aspergillus niger (wild or parental
strains) were isolated from different rotted sources.
Media:
I. Potato-dextrose agar medium (PDA) was used for isolation.
Strain of A. niger were grown on PDA medium, incubated
at 28°C for 7 days, this medium also used for purification
and maintenance the strains.
II. The indicator medium used for screening the strains and
citric acid production as described earlier (Chopra et al.,
1983). This medium contained (g/l) glucose 20.0, KH2
Po4,1.0; Mg So4, 7H2O, 0.25; NH4 No3, 2.50; pH, 5.5;
bromocresol purple (0.4% in alcohol), 10ml; agar agar,
20.0; the basal liquid medium used for production of citric
acid refer to the indicator medium minus carbon source,
agar-agar and the bromocresol purpule.
Inoculum preparation:
Spore suspension was prepared from 7 days old culture of
parental A. niger strains with sterilized water with shaking
vigorously for 1 min, the spore density was adjusted to
- 24 -
107spores/ml of the suspension (Mourya & Jauhri 2000 and
Ikram et al., 2004).
Carbohydrate by-products:
Maize straw and potato solid wastes were cut to small
pieces, sugar beet pulp was grounded to very small pieces (Baig
et al., 2004) and molasses diluted by water (1:3). These substrates
used without any treatment for growing all the strains alone at
different concentrations i.e. 5,10,15,20 & 25%, without any
additions, and also used for growing all the strains as carbon
source instead of glucose in basal liquid medium.
Cultivation:
A niger strains allowed to grow in 100ml fermentation
medium dispensed in 250ml Erlenmeyer flasks. 1 ml of 107 spores
were used to inoculate the flasks, then incubated at 30°C. Samples
were taken at different time intervals i.e. 6 and 12 days and tested
for citric acid production (Mourya & Jauhri 2000 and Ikram et
al., 2004).
Exposure of selected parental strains of Aspergillus niger to
uv light irradiation and γγγγ-rays:
A spore suspension was prepared (107 spores/ml), 0.1ml of
spore suspension was exposed to uv irradiation (wavelength 320
nm) at a distance of 30cm with exposure times of 2, 5, 10, 15, 20,
- 25 -
25 & 30 min. The uv lamp was used in dark environment in order
to avoid the repair mechanism that could invert the mutagenic
effects (Zaha 2003 and Rodrigues et al., 2010).
The spore suspension was also exposed to different does of
gamma rays i.e., 25, 50, 100, 200 Gy by Co-60 gamma indicator
(dose rate 11.1Gy/min) (Golubtsova et al., 1978, 1976&1972
and El-Batal et al., 1995) located at cyclotorn at Nuclear
Research Center, Inshas, Egypt.
Isolation of obtained strains (treated isolates):
Spore suspensions (0.1ml) were subjected to uv and gamma
rays, then plated on the indicator medium. The plates were
wrapped in carbon paper and incubated in dark at 30°C for a
period of 48 h. Colonies appeared in plates of each treatment were
obtained and tested for citric acid production (Mourya and
Jauhri 2000).
Assay of citric acid:
Citric acid was estimated gravimetrically, using pyridine-
acetic anhydride method as reported by (Marrier & Boulet, 1958
and Ikram et al., 2004). One ml of the culture filtrate along with
1.3ml of pyridine was added in the test tube and shacked then
5.70ml of acetic anhydride was added in the test tube. The test
tube was placed in a water bath for 30min. The optical density
- 26 -
was measured on a spectrophotometric (405 nm) and citric acid
contents of the sample was estimated with reference (run parallel,
replacing 1.0 ml of the culture filtrate with distilled water) to the
standard. Data recorded are the mean of three replicates ±
standard deviation.
0.0 0.2 0.4 0.6 0.8 1.0
0.1
0.2
0.3
0.4
0.5
0.6
Fig.(1): Standard curve citric acid.
Optical density
Conc. of citric acid, (mg/ml)
Random Amplified Polymorphic DNA Polymerase Chain
Reaction (RAPD-PCR):
There were a number of standard protocols by which
specific DNA sequences can be subjected to successive
amplification. One of the most reliable techniques currently used
in molecular biology is DNA amplification by PCR, which is a
- 27 -
procedure that allows rapid detection of the presence or absence
of a target DNA sequence in any genetic material. In this
technique, DNA is amplified in vitro by a series of polymerization
cycles consisting of three temperature-dependent steps
(Denaturation, annealing and extension) resulting in target DNA
amplification (Mullis et al., 1986 and Rychlik et al., 1990).
Nearly ten years ago, anew genetic assay was developed
indepently by two different laboratories (Welsh & Neclellend,
1990 and Williams et al., 1991). This procedure, which we have
called the RAPD assay, detects nucleotide sequence
polymorphisms in a DNA amplification-based as say using only a
single primer of arbitrary nucleotide sequence. In this reaction, a
single species of primer binds to the genomic DNA at two
different sites on opposite strands of the DNA template.
DNA was isolated using Maxweld 16 (promega) instrument
according to the manufacture instructions.
RAPD-PCR
Reactions were performed in a total volume 50µl reaction
buffer (100 mM KC1, 100 mm Tris HC1 pH 8.3) 3.0mm MgCl2,
200mm dNTPs (Promega Biotec. Inc.) 50p/mole primers and 0.2
µl Taq Polymerase (Hot Start). This reaction was added to 0.1µl
genomic DNA. All reactions tubes, pipette tips, micro pestles and
- 28 -
water were irradiated with uv light to destroy possible
contaminating surface DNA (Ou et al., 1991). Irradiation
treatment was 20 minutes at 2.5cm from the bulbs of a Gene
linker (Biorad, inc.) uv light source.
Tubes contain mixes were placed in a thermcycler (Perkin-
Elmer 2400) and DNA was amplified using the following
temperature cycle modified from (Black et al., 1992).
Preparation of PCR Reactions
The reactions were carried out in a volume of 50µl
containing 250ng of genomic DNA template, 2.5mM MgCl2,
1- A master mixture for 12 reactions (10 PCR wells) was
prepared in 1.5 ml microcentrifuge tube, so that each reaction
contained.
Component Amount for one PCR reaction
H2O 35.1µl
10 reaction buffer 5µl
DNTPs mix (100 mM) 1µl
2- An aliquot of 43.6 µl master mix was dispensed in each PCR
tube (o.5ml).
3- 5 µl of the primer (10 p/mole) were added to each tube.
4- 1 µl of the template (250 ng) was added.
5- 0.2 µl of Hot start Taq (1.25 Unit) was added.
- 29 -
PCR Program and temperature profile
Amplification of the DNA was performed by placing the
tubes containing the reactions in a Perkin Elmer thermal cycler
2400.
RAPD PCR performed in 50 ul reaction volumes for 30
cycles. After the reaction mixture was mixed with DNA loading
buffer and electrophoresed on 1% Agarose gel.
Temperature profile
Step 0: 94°C 5 min
Step l: 94°C 40 sec
Step 2: 36°C 1 min
Step 3: 72 °C 2 min
Step 4: 72 °C 7 min
Step 5: 4°C Hold
Protein electrophoresis
Sodium dodecyl sulfate (SDS) - Polyaciylamide gel
It was carried out at Agricultural Research Center,
Agricultural Genetic Engineering Research Institute.
Discontinuous Polyacrylamide gels consist of a resolving or
separating (lower) gel and stacking (upper) gel. The stacking gel
acts to large sample volumes, resulting in better band resolution
than, if possible, using the same volumes on a gel without a
stacJc. Gels were prepared according to (Laemmli, 1970).
- 30 -
Preparation of separating gel (10%)
Distilled water 4 ml
1.5 MTris-HCI 2.50 ml
10% SDS 100µl
Acrylamide/bis (30%) 3.33 µl
10% APS 50 µl
TEMED 5 µl
Volume required to completely fill gel sandwich may be adjusted
depending on application.
Preparation of stacking gel (4.0%)
Distilled water 3 ml
0.5MTris-HCI 1.25ml
10%SDS 50 µl
Acrylamide/bis (30%) 670 µl
10% APS 25 µl
TEMED 5 µl
To prepare monomer solution of both separating and
stacking gels, combine all reagents, except the TEMED and
ammonium persulphate (APS), and degas under vacuum for 10
minutes. Add the two catalysis just prior to casting the gels. The
amount of stacking gel may be adjusted depending on the height
of the separating gel. Higher TEMBD concentration for faster
- 31 -
polymerization is required for the stacking gel because of the
inhibitory effect of atmospheric oxygen associated with the comb.
Stock solutions
Acrylamide/bis (30%)
Acrylamide 29.2 g
N-N-bis-methylene-acrylami 0.8 g
Make to 100 ml with distilled water. Filter and store at 4°C in
dark (30 days maximum).
1.5 M Tris-HCI
Trisbase 18.15g Distilled water 60 ml
Adjust pH 8.8 with IN HC1. Complete the volume to 100 ml with
distilled water and store at4°C.
0.5M Tris-HCI
Tris base 6g
Distilled water 60 ml
Adjust to pH 6.8 with IN HC1, complete the volume to 100
ml with distilled water and store at 4°C.
10% SDS
Dissolve 10 g SDS in warm water with gentle stirring and
bring the volume to 100 ml with distilled water.
- 32 -
10 % ammonium persulphate (APS)
Dissolve 100 mg APS in 1 ml distilled water.
Ammonium sulfate {(NH4)2SO4=132.13 MWT} Provided by
BDH laboratory supplies Poole, BH 15 LTD, England
{Tel: (01202) 669700}.
Treatment buffer (62.5 mM Tris, 20% Glycerol, 2% SDS &
5% B-ME)
Distilled water 4.0 ml
0.5MTris-HCl 1.0ml
Glycerol 0.8 ml
10% SDS 1.6ml
B-ME 0.4 ml
0.05% (W/V) Bromophenol blue 0.2 ml
5% Tank buffer (IX = 25 mM Tris, 192mM glycine and 0,1%
SDS)
Tris base 15 g
Glycine 72 g
SDS 5 g
Complete to one liter with distilled water and store at 4°C.
The working solution is IX with pH 8.3. Staining solution
Coomassie blue R-250 0.1 %
Methanol 40 %
Glacial acetic acid 10%
- 33 -
Destaining solution
Glacial acetic 10%
Methanol 40%
Completed to 100m of distilled water
Protein banding patterns
Preparation and solubilization of the total proteins
Total proteins of the four isolates of Aspergillus niger were
analyzed by SDS-PAGE. Pellets were collected by centrifugation
at 12000 rpm at 4°C, washed once with distilled water and then
with 1 ml of ImM NaCl containing 5 mM EDTA. Proteins were
separated on the basis of molecular weight by Sodium Dodecyl
Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
(Laemmli,1970), then heated in the present of low molecular
weight thiol (2-mercaptoethanol) and SDS denatured total cellular
protein from vegetative or sporulated cells. One volume of the
cell suspension was mixed with one volume of 2X treatment
buffer (0.25M Tris-HCL PH6.8, 4% SDS, 20% glycerol and 10%
2-mercaptoethanol) and boiled in a water bath for 90 seconds then
quickly transferred to ice water and kept until loading the gel.
Protein electrophoresis
The gel apparatus was assembled and the lower and upper
chambers were filled with the tank buffer. A drop of
- 34 -
Bromophenol blue was added as a tracking dye. A Hamilton
syringe was used to load equal amounts of proteins (25 M1) in
each well. High range molecular weight protein marker from Bio-
Rad was used. Electrophoresis was carried out at about 100 volts
in Ix Tris/glycine- SDS-running buffer. After electrophoresis, the
gel was stained in 50ml of staining solution (0.125% Coomassie
blue R-250, 50% methanol and 10% acetic acid). The gel was
placed between two sheets of cellophane membrane and dried on
gel drier for 2hrs and photographed.
Preparation and casting of the SDS-PoIyacrylamide Gel
The gels were prepared from monomer solution of 30%
Acrylamide and 2.7% Bis-Acrylamide. The gels were prepared as
separating gel containing 10% acrylamide monomer and stacking
gel 4%. Ammonium persulphate and TEMED were used as
initiators for cross-linking and polymerization. The details of the
solution are mentioned in the material section. The components of
the separating gel solution were placed in 125ml side arm vacuum
flask, stoppered flask and apply vacuum for several minutes to
insure suction of oxygen from the solution. Ammonium
persulphate and TEMED were added and the flask gently swirled
to mix the solutions being careful not to generate babbles. The
solution was pipetted into the assembled vertical slab gel unit in
the casting mode to level 1.5cm from the top. N-butanol was
- 35 -
layered on the top of the solution. The gel was left to polymerize
at room temperature for two hours. Prior to addition of stacking
gel, the N-butanol was poured from the surface of the gel and the
surface was washed once with overlay buffer. The 4% stacking
gel was prepared by mixing its components as indicated in
materials section, degassing under vacuum, adding TEMED and
ammonium persulphate and gently swirling the mixture. Stacking
solution was added to the top of the separating gel, combs were
inserted and the gel was allowed to polymerize for at least half an
hour. After polymerization the combs were removed slowly from
the gel and each well was rinsed with tank buffer using a
Hamilton syringe.
- 36 -
- 37 -
Results
Twenty strains of Aspergillus niger were isolated from
different sources (Table 1) they were screened for their capacity
to produce citric acid on indicator medium and different
carbohydrate by-products:
1. Production of citric acid by Aspergillus niger strains on
indicator medium:
Data recorded in Table (2) showed that all the strains under
the study were able to produce citric acid on indicator medium as
indicated by the diameter of the clear zone (mm) and production
in different quantities at different time intervals i.e. 4, 8 and 12
days. Data showed that the best incubation period for production
was 12 days for all isolates. Maximum production on indicator
medium after 12 days were 0.95, 0.94 & 0.99mg/ml for A1, A4 &
A5 respectively (Figs. 2& 3) while the lowest production recorded
in this medium were 0.74, 0.71, 0.68 & 0.62 mg/ml for A8, A16,
A18 & A19 respectively (Figs. 2 & 4).
- 38 -
Table (1): Source of isolation of twenty strains of A. niger.
Strains of A. niger Source of rotted material
A1 Maize grains
A2 Chesse
A3 Peanut
A4 Banana
A5 Apple
A6 Potato
A7 Bread
A8 Phylloplane of Solanum persicum
A9 Orange
A10 Peach
A11 Bagasse
A12 Tomato
A13 Peach
A14 Pickles
A15 Yoghurt
A16 Garlic
A17 Lemon
A18 Potato
A19 Tomato sauce
A20 Bread
- 39 -
Table (2): Diameter of clear zone (mm) and production of
citric acid (mg/ml) by different strains of Aspergillus
niger using indicator medium at different time
intervals.
Diameter of clear zone (mm) Citric acid production (mg/ml)
Isolates
4 days 8 days 4 days 8 days 12 days
A1 50 90 0.38±±±±0.04 0.67±±±±0.03 0.95±±±±0.08
A2 35 55 0.25±±±±0.09 0.56±±±±0.06 0.81±±±±0.09
A3 45 80 0.36±±±±0.07 0.65±±±±0.10 0.91±±±±0.13
A4 48 85 0.38±±±±0.03 0.66±±±±0.05 0.94±±±±0.07
A5 51 92 0.39±±±±0.06 0.68±±±±0.08 0.99±±±±0.12
A6 25 45 0.21±±±±0.07 0.52±±±±0.04 0.76±±±±0.10
A7 27 47 0.24±±±±0.09 0.53±±±±0.09 0.77±±±±0.06
A8 30 38 0.20±±±±0.09 0.50±±±±0.09 0.74±±±±0.04
A9 37 70 0.31±±±±0.12 0.60±±±±0.11 0.88±±±±0.11
A10 40 75 0.34±±±±0.04 0.62±±±±0.07 0.88±±±±0.08
A11 36 55 0.26±±±±0.05 0.56±±±±0.06 0.82±±±±0.06
A12 35 60 0.27±±±±0.03 0.57±±±±0.06 0.84±±±±0.07
A13 23 42 0.20±±±±0.09 0.51±±±±0.05 0.76±±±±0.09
A14 38 65 0.29±±±±0.10 0.59±±±±0.13 0.85±±±±0.09
A15 37 65 0.29±±±±0.11 0.59±±±±0.06 0.86±±±±0.11
A16 22 30 0.20±±±±0.09 0.48±±±±0.08 0.71±±±±0.04
A17 39 72 0.32±±±±0.09 0.61±±±±0.04 0.90±±±±0.07
A18 25 30 0.19±±±±0.07 0.47±±±±0.09 0.68±±±±0.03
A19 17 22 0.17±±±±0.03 0.45±±±±0.05 0.62±±±±0.05
A20 38 62 0.26±±±±0.03 0.58±±±±0.07 0.85±±±±0.08
- 40 -
- 41 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
citric acid production (mg/ml)
A1 A4 A5
A. niger isolates
4 days
8 days
12days
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
citric acid production (mg/ml)
A 8 A 16 A 18 A 19
Weak isolates
4 days
8 days
12days
Fig. (3): Maximum Citric acid production by A. niger isolates (A1, A4 &
A5) on indicator medium at different time intervals.
Fig. (4): Weakest production of citric acid by four isolates (A8, A16, A18
& A19) on indicator medium at different time intervals.
- 42 -
2. Production of citric acid by Aspergillus niger strains on
maize straw after 12 days incubation:
Table (3) showed citric acid production (mg/ml) by A.
niger strains when allowed to grow on different concentrations of maize straw i.e., 5, 10, 15, 20 & 25% without any additions after 12 days incubation. Maximum production were 0.68, 0.64 & 0.70 mg/ml by A1, A4 & A5 respectively at 25% concentration. It can be noticed from the table that increasing the concentration of maize straw led to increase citric acid production by the isolates. The best concentration for production was 25%. The lowest production at 25% maize straw obtained by A8, A16, A18 & A19, the production were 0.48, 0.47, 0.47 & 0.46 mg/ml respectively (Table 3 & Fig. 5).
Data in table (4) showed citric acid production (mg/ml) by Aspergillus niger isolates when allowing to grow on basal medium amended with the previous mentioned concentrations of maize straw after 12 days incubation. Maximum production recorded were 0.73, 0.70 & 0.84mg/ml by A1, A4 & A5 respectively at 25% concentration (Fig. 6). It can be noticed that the production of these isolates growing on a basal medium amended with maize straw was more than that produced by isolates grown on maize straw only. Also increasing substrate concentration led to increase citric acid production for all Aspergillus niger isolates. Weak isolates (A8, A16, A18 & A19) which grown on basal medium amended with maize straw showed rarely similar production levels to that of straw alone.
- 43 -
Table (3): Citric acid production (mg/ml) by different isolates
of A. niger grown on different concentrations of
maize straw after 12 days incubation.
Citric acid production (mg/ml)
Maize straw (%) Strain No.
5 10 15 20 25
A1 0.26±±±±0.07 0.31±±±±0.02 0.45±±±±0.07 0.55±±±±0.05 0.68±±±±0.04
A2 0.19±±±±0.02 0.25±±±±0.05 0.38±±±±0.05 0.40±±±±0.03 0.51±±±±0.07
A3 0.24±±±±0.06 0.28±±±±0.03 0.40±±±±0.06 0.53±±±±0.05 0.63±±±±0.03
A4 0.22±±±±0.08 0.30±±±±0.09 0.44±±±±0.03 0.54±±±±0.11 0.64±±±±0.05
A5 0.27±±±±0.03 0.33±±±±0.08 0.46±±±±0.07 0.58±±±±0.09 0.70±±±±0.09
A6 0.22±±±±0.05 0.24±±±±0.06 0.33±±±±0.09 0.39±±±±0.07 0.51±±±±0.08
A7 0.21±±±±0. 10 0.27±±±±0.04 0.34±±±±0.10 0.41±±±±0.10 0.53±±±±0.08
A8 0.16±±±±0.09 0.21±±±±0.07 0.31±±±±0.12 0.35±±±±0.09 0.48±±±±0.06
A9 0.20±±±±0.07 0.24±±±±0.03 0.34±±±±0.07 0.43±±±±0.05 0.59±±±±0.11
A10 0.20±±±±0.07 0.25±±±±0.09 0.37±±±±0.05 0.50±±±±0.06 0.62±±±±0.12
A11 0.20±±±±0.06 0.24±±±±0.12 0.37±±±±0.09 0.40±±±±0.11 0.53±±±±0.10
A12 0.20±±±±0.03 0.24±±±±0.04 0.31±±±±0.08 0.41±±±±0.05 0.55±±±±0.04
A13 0.17±±±±0.03 0.22±±±±0.05 0.32±±±±0.11 0.38±±±±0.07 0.50±±±±0.05
A14 0.23±±±±0.12 0.24±±±±0.11 0.33±±±±0.09 0.44±±±±0.04 0.56±±±±0.07
A15 0.23±±±±0.07 0.23±±±±0.08 0.32±±±±0.09 0.44±±±±0.08 0.56±±±±0.08
A16 0.15±±±±0.09 0.20±±±±0.04 0.29±±±±0.08 0.32±±±±0.09 0.47±±±±0.04
A17 0.21±±±±0.06 0.23±±±±0.07 0.35±±±±0.08 0.47±±±±0.05 0.61±±±±0.03
A18 0.15±±±±0.09 0.21±±±±0.05 0.30±±±±0.04 0.33±±±±0.07 0.47±±±±0.03
A19 0.13±±±±0.11 0.17±±±±0.04 0.27±±±±0.07 0.30±±±±0.07 0.45±±±±0.05
A20 0.20±±±±0.03 0.22±±±±0.03 0.30±±±±0.09 0.44±±±±0.03 0.56±±±±0.07
- 44 -
Table (4): Citric acid production (mg/ml) by different isolates
of A. niger growing on basal medium amended
with different concentrations of maize straw after
12 days incubation.
Citric acid production (mg/ml)
Maize straw (%) Strain No.
5 10 15 20 25
A1 0.35±±±±0.07 0.48±±±±0.05 0.61±±±±0.3 0.70±±±±0.08 0.73±±±±0.04
A2 0.28±±±±0.04 0.39±±±±0.08 0.45±±±±0.07 0.51±±±±0.06 0.56±±±±0.03
A3 0.31±±±±0.07 0.51±±±±0.09 0.57±±±±0.05 0.60±±±±0.03 0.69±±±±0.07
A4 0.35±±±±0.06 0.45±±±±0.12 0.59±±±±0.04 0.65±±±±0.03 0.70±±±±0.09
A5 0.36±±±±0.06 0.53±±±±0.05 0.62±±±±0.04 0.73±±±±0.07 0.84±±±±0.11
A6 0.24±±±±0.09 0.33±±±±0.07 0.42±±±±0.03 0.50±±±±0.09 0.53±±±±0.08
A7 0.23±±±±0.12 0.31±±±±0.06 0.42±±±±0.08 0.49±±±±0.09 0.55±±±±0.05
A8 0.21±±±±0.08 0.30±±±±0.09 0.38±±±±0.06 0.41±±±±0.03 0.48±±±±0.03
A9 0.29±±±±0.08 0.39±±±±0.09 0.48±±±±0.07 0.53±±±±0.08 0.61±±±±0.07
A10 0.31±±±±0.04 0.41±±±±0.08 0.53±±±±0.09 0.57±±±±0.09 0.67±±±±0.06
A11 0.28±±±±0.03 0.38±±±±0.04 0.47±±±±0.06 0.54±±±±0.04 0.57±±±±0.09
A12 0.27±±±±0.07 0.34±±±±0.11 0.44±±±±0.05 0.51±±±±0.04 0.58±±±±0.08
A13 0.21±±±±0.09 0.30±±±±0.06 0.37±±±±0.05 0.42±±±±0.08 0.51±±±±0.03
A14 0.20±±±±0.03 0.32±±±±0.06 0.45±±±±0.04 0.53±±±±0.07 0.59±±±±0.09
A15 0.21±±±±0.03 0.33±±±±0.09 0.45±±±±0.11 0.53±±±±0.06 0.59±±±±0.04
A16 0.17±±±±0.05 0.22±±±±0.04 0.33±±±±0.03 0.40±±±±0.06 0.48±±±±0.11
A17 0.35±±±±0.08 0.40±±±±0.03 0.54±±±±0.03 0.53±±±±0.03 0.65±±±±0.010
A18 0.18±±±±0.06 0.23±±±±0.05 0.35±±±±0.13 0.40±±±±0.07 0.48±±±±0.04
A19 0.14±±±±0.09 0.19±±±±0.05 0.30±±±±0.05 0.33±±±±0.09 0.46±±±±0.07
A20 0.28±±±±0.11 0.35±±±±0.07 0.44±±±±0.12 0.51±±±±0.11 0.58±±±±0.03
- 45 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger growing on 25% maize straw
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger growing on basal medium amended with 25% of maize straw
Fig. (5): Citric acid production (mg/ml) by different isolates of A. niger
growing on 25% maize straw after 12 days incubation.
Fig. (6): Citric acid production (mg/ml) by different isolates of A. niger
growing on basal medium amended with 25% maize straw
after 12 days incubation.
- 46 -
3. Production of citric acid by Aspergillus niger isolates on
potato solid wastes (peels) after 12 days incubation:
Increasing concentration of potato solid wastes led to
increase citric acid production for all Aspergillus niger strains
(tables 5 & 6). The best concentration for production was 25%,
maximum production were obtained by A1 & A4, A5 after 12
days incubation. Production of these isolates on 25% potato solid
wastes were 0.75, 0.74 & 0.76mg/ml respectively (table 5 & Fig.
7) while the yield on basal medium amended with 25% potato
solid wastes were 0.94, 0.91 & 0.98mg/ml respectively (table 6 &
Fig. 8). The production for all the strains on the basal medium
amended with potato solid wastes showed an increase over that
with potato solid wastes only. Lowest production recorded with
A8, A16, A18 & A19 in case of growing on both potato solid wastes
only and on basal medium amended with that carbohydrate by
product as shown from the two tables (5&6).
4. Production of citric acid by Aspergillus niger isolates on
sugar beet pulp after 12 days incubation:
The highest production obtained by A1, A4 & A5, when
allowing to grow on sugar beet pulp & also on basal medium
amended with that carbohydrate by- product (tables 8 & 9). The
production on 25% sugar beet pulp were: 1.10, 1.05 & 1.08
mg/ml respectively (table 8 & Fig. 9). While the production on
basal medium amended with sugar beet pulp were: 1.56, 1.46 &
1.52 mg/ml respectively (Fig. 10).
- 47 -
Table (5): Citric acid production (mg/ml) by different isolates
of A. niger grown on different concentrations of
potato solid wastes after 12 days incubation.
Citric acid production (mg/ml)
Potato solid wastes (%) Strain No.
5 10 15 20 25
A1 0.37±±±±0.11 0.48±±±±0.08 0.55±±±±0.04 0.67±±±±0.07 0.75±±±±0.06
A2 0.23±±±±0.04 0.35±±±±0.07 0.44±±±±0.12 0.53±±±±0.10 0.60±±±±0.08
A3 0.33±±±±0.09 0.46±±±±0.10 0.54±±±±0.02 0.64±±±±0.04 0.71±±±±0.05
A4 0.35±±±±0.03 0.47±±±±0.07 0.54±±±±0.08 0.65±±±±0.04 0.74±±±±0.09
A5 0.38±±±±0.07 0.49±±±±0.09 0.57±±±±0.03 0.68±±±±0.04 0.76±±±±0.07
A6 0.22±±±±0.11 0.31±±±±0.08 0.48±±±±0.07 0.57±±±±0.06 0.59±±±±0.04
A7 0.22±±±±0.07 0.32±±±±0.05 0.47±±±±0.03 0.56±±±±0.09 0.59±±±±0.05
A8 0.21±±±±0.08 0.28±±±±0.09 0.42±±±±0.04 0.51±±±±0.03 0.56±±±±0.03
A9 0.28±±±±0.06 0.44±±±±0.07 0.51±±±±0.08 0.57±±±±0.04 0.65±±±±0.07
A10 0.30±±±±0.06 0.45±±±±0.05 0.53±±±±0.03 0.61±±±±0.04 0.68±±±±0.03
A11 0.23±±±±0.09 0.29±±±±0.03 0.44±±±±0.06 0.57±±±±0.03 0.61±±±±0.09
A12 0.24±±±±0.04 0.40±±±±0.03 0.47±±±±0.07 0.59±±±±0.05 0.63±±±±0.10
A13 0.21±±±±0.07 0.30±±±±0.06 0.45±±±±0.09 0.51±±±±0.12 0.57±±±±0.9
A14 0.27±±±±0.03 0.42±±±±0.07 0.49±±±±0.09 0.58±±±±0.14 0.64±±±±0.11
A15 0.26±±±±0.03 0.42±±±±0.09 0.47±±±±0.12 0.57±±±±0.08 0.64±±±±0.04
A16 0.20±±±±0.11 0.25±±±±0.07 0.43±±±±0.03 0.50±±±±0.09 0.55±±±±0.07
A17 0.29±±±±0.09 0.45±±±±0.06 0.52±±±±0.08 0.60±±±±0.05 0.67±±±±0.03
A18 0.20±±±±0.03 0.26±±±±0.09 0.43±±±±0.06 0.50±±±±0.07 0.53±±±±0.06
A19 0.18±±±±0.04 0.22±±±±0.08 0.40±±±±0.09 0.48±±±±0.03 0.51±±±±0.05
A20 0.25±±±±0.05 0.42±±±±0.70 0.49±±±±0.04 0.57±±±±0.03 0.64±±±±0.07
- 48 -
Table (6): Citric acid production (mg/ml) by different isolates of A. niger
grown on basal medium amended with different
concentrations of potato solid wastes after 12 days
incubation.
Citric acid production (mg/ml)
Potato solid wastes (%) Strain No.
5 10 15 20 25
A1 0.36±±±±0.05 0.58±±±±0.04 0.77±±±±0.07 0.85±±±±0.09 0.94±±±±0.03
A2 0.29±±±±0.07 0.48±±±±0.08 0.51±±±±0.06 0.55±±±±0.11 0.69±±±±0.09
A3 0.34±±±±0.03 0.56±±±±0.08 0.74±±±±0.04 0.78±±±±0.12 0.88±±±±0.10
A4 0.34±±±±0.08 0.57±±±±0.05 0.76±±±±0.07 0.80±±±±0.06 0.91±±±±0.08
A5 0.37±±±±0.06 0.58±±±±0.04 0.77±±±±0.08 0.89±±±±0.10 0.98±±±±0.09
A6 0.26±±±±0.03 0.37±±±±0.09 0.46±±±±0.08 0.54±±±±0.03 0.65±±±±0.07
A7 0.26±±±±0.07 0.38±±±±0.08 0.48±±±±0.04 0.56±±±±0.04 0.66±±±±0.03
A8 0.25±±±±0.10 0.36±±±±0.12 0.43±±±±0.03 0.54±±±±0.07 0.62±±±±0.08
A9 0.30±±±±0.08 0.41±±±±0.04 0.65±±±±0.03 0.69±±±±0.06 0.79±±±±0.05
A10 0.32±±±±0.07 0.43±±±±0.05 0.69±±±±0.06 0.75±±±±0.06 0.84±±±±0.12
A11 0.29±±±±0.03 0.47±±±±0.09 0.51±±±±0.09 0.55±±±±0.09 0.68±±±±0.09
A12 0.30±±±±0.07 0.46±±±±0.11 0.58±±±±0.04 0.63±±±±0.03 0.75±±±±0.04
A13 0.28±±±±0.05 0.37±±±±0.04 0.44±±±±0.03 0.54±±±±0.09 0.63±±±±0.03
A14 0.33±±±±0.04 0.48±±±±0.05 0.61±±±±0.07 0.65±±±±0.04 0.77±±±±0.05
A15 0.33±±±±0.03 0.49±±±±0.06 0.62±±±±0.09 0.66±±±±0.07 0.78±±±±0.05
A16 0.24±±±±0.03 0.37±±±±0.08 0.41±±±±0.09 0.52±±±±0.07 0.60±±±±0.07
A17 0.31±±±±0.08 0.51±±±±0.09 0.66±±±±0.10 0.70±±±±0.10 0.81±±±±0.11
A18 0.25±±±±0.09 0.38±±±±0.03 0.41±±±±0.013 0.51±±±±0.08 0.60±±±±0.09
A19 0.23±±±±0.09 0.35±±±±0.03 0.38±±±±0.04 0.49±±±±0.07 0.58±±±±0.05
A20 0.30±±±±0.11 0.43±±±±0.07 0.65±±±±0.05 0.71±±±±0.07 0.80±±±±0.06
- 49 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on 25% of potato solid wastes
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on basal medium amended w ith 25% of potato
solid wastes
Fig. (7): Citric acid production (mg/ml) by different isolates of A. niger growing
on 25% of potato solid wastes after 12 days incubation.
Fig. (8): Citric acid production (mg/ml) by different isolates of A. niger growing
on basal medium amended with 25% of potato solid wastes after 12
days incubation.
- 50 -
Increasing substrate concentrations led to increase the production
of the isolates, the best concentration for production under the study
was also at 25% in this carbohydrate by-product. Lowest production at
25% concentration on both sugar beet pulp and basal medium amended
with sugar beet pulp obtained by A8, A16, A18, A19. The productions of
these isolates on sugar beet pulp were 0.91, 0.72. 0.69, & 0.65mg/ml
while on basal medium amended with sugar beet pulp were: 0.94, 0.90,
0.85 & 0.82mg/ml respectively (table 8 & Fig. 10). It can be noticed that
citric acid production of the isolates on the basal medium amended with
sugar beet pulp was more than that if the isolates grown on sugar beet
pulp alone.
5. Production of citric acid by Aspergillus niger isolates on
molasses after 12 days incubation:
Increasing molasses concentration under the study led to increase
the production of citric acid for all the isolates when allowing to grow on
both molasses only and on basal medium amended with molasses, the
best concentration for production was 25% (Tables 9 & 10). Highest
citric acid production recorded with: A1, A4 and A5 isolates in case of
growing on molasses only and also on basal medium amended with
molasses. Production of these isolates on 25% molasses only were 0.94,
0.84 and 0.86 mg/ml respectively (table 9). While on basal medium
amended with 25% molasses the production was 1.17, 1.04 & 1.15 mg/ml
respectively (table 10) while the lowest citric acid production obtained
by isolates A8, A16, A18 & A19, the production on 25% molasses were:
0.72, 0.67, 0.65 & 0.59 mg/ml respectively (Fig. 11), while the production
on basal medium amended with 25% molasses were: 0.80, 0.78, 0.76 &
0.65 mg/ml respectively (Fig. 12).
- 51 -
Table (7): Citric acid production (mg/ml) by different isolates
of A. niger growing on different concentrations of
sugar beet pulp after 12 days incubation.
Citric acid production (mg/ml)
Sugar beet pulp (%) Strain No.
5 10 15 20 25
A1 0.50±±±±0.12 0.68±±±±0.04 0.84±±±±0.05 0.91±±±±0.07 1.10±±±±0.09
A2 0.37±±±±0.06 0.52±±±±0.09 0.66±±±±0.04 0.79±±±±0.03 0.90±±±±0.10
A3 0.39±±±±0.07 0.63±±±±0.07 0.84±±±±0.08 0.91±±±±0.11 1.00±±±±0.04
A4 0.48±±±±0.09 0.69±±±±0.03 0.86±±±±0.06 0.93±±±±0.05 1.05±±±±0.03
A5 0.49±±±±0.04 0.70±±±±0.09 0.89±±±±0.09 0.92±±±±0.03 1.08±±±±0.08
A6 0.35±±±±0.03 0.60±±±±0.07 0.72±±±±0.12 0.83±±±±0.04 0.95±±±±0.11
A7 0.38±±±±0.13 0.65±±±±0.06 0.77±±±±0.10 0.88±±±±0.03 0.98±±±±0.09
A8 0.34±±±±0.05 0.48±±±±0.08 0.64±±±±0.03 0.74±±±±0.04 0.91±±±±0.07
A9 0.41±±±±0.06 0.53±±±±0.07 0.70±±±±0.07 0.83±±±±0.03 1.00±±±±0.09
A10 0.44±±±±0.09 0.58±±±±0.03 0.76±±±±0.07 0.80±±±±0.04 1.03±±±±0.08
A11 0.39±±±±0.06 0.57±±±±0.07 0.64±±±±0.10 0.82±±±±0.03 0.95±±±±0.03
A12 0.41±±±±0.09 0.59±±±±0.11 0.70±±±±0.18 0.85±±±±0.07 0.99±±±±0.04
A13 0.32±±±±0.03 0.45±±±±0.04 0.60±±±±0.09 0.73±±±±0.08 0.84±±±±0.07
A14 0.42±±±±0.04 0.63±±±±0.07 0.74±±±±0.03 0.89±±±±0.04 1.02±±±±0.09
A15 0.40±±±±0.08 0.60±±±±0.03 0.72±±±±0.03 0.84±±±±0.09 1.00±±±±0.06
A16 0.35±±±±0.11 0.44±±±±0.12 0.56±±±±0.09 0.65±±±±0.04 0.72±±±±0.07
A17 0.41±±±±0.07 0.60±±±±0.03 0.74±±±±0.10 0.87±±±±0.03 0.94±±±±0.04
A18 0.30±±±±0.04 0.40±±±±0.06 0.52±±±±0.05 0.64±±±±0.09 0.69±±±±0.10
A19 0.31±±±±0.03 0.38±±±±0.06 0.53±±±±0.07 0.60±±±±0.06 0.65±±±±0.09
A20 0.42±±±±0.05 0.59±±±±0.09 0.74±±±±0.07 0.89±±±±0.07 0.95±±±±0.11
- 52 -
Table (8): Citric acid production (mg/ml) by different isolates
of A. niger growing on basal liquid medium
amended with different concentrations of sugar
beet pulp after 12 days incubation.
Citric acid production (mg/ml)
Sugar beet pulp (%) Strain No.
5 10 15 20 25
A1 0.55±±±±0.06 0.75±±±±0.09 0.97±±±±0.07 1.15±±±±0.05 1.56±±±±0.03
A2 0.43±±±±0.08 0.69±±±±0.03 0.88±±±±0.04 0.93±±±±0.07 1.28±±±±0.03
A3 0.45±±±±0.09 0.75±±±±0.03 0.91±±±±0.09 0.97±±±±0.12 1.40±±±±0.04
A4 0.51±±±±0.05 0.72±±±±0.03 0.92±±±±0.04 1.06±±±±0.03 1.46±±±±0.05
A5 0.53±±±±0.11 0.73±±±±0.05 0.93±±±±0.09 1.09±±±±0.07 1.52±±±±0.06
A6 0.40±±±±0.09 0.66±±±±0.09 0.78±±±±0.03 0.89±±±±0.04 1.02±±±±0.09
A7 0.42±±±±0.09 0.71±±±±0.07 0.80±±±±0.07 0.90±±±±0.06 1.08±±±±0.06
A8 0.38±±±±0.03 0.57±±±±0.04 0.72±±±±0.06 0.80±±±±0.09 0.94±±±±0.07
A9 0.47±±±±0.09 0.69±±±±0.07 0.81±±±±0.03 0.92±±±±0.04 1.24±±±±0.08
A10 0.50±±±±0.04 0.73±±±±0.03 0.87±±±±0.06 0.96±±±±0.07 1.33±±±±0.09
A11 0.45±±±±0.08 0.62±±±±0.07 0.74±±±±0.03 0.90±±±±0.06 1.11±±±±0.04
A12 0.49±±±±0.11 0.64±±±±0.08 0.77±±±±0.09 0.92±±±±0.04 1.20±±±±0.07
A13 0.37±±±±0.12 0.53±±±±0.05 0.66±±±±0.014 0.84±±±±0.06 0.92±±±±0.05
A14 0.50±±±±0.06 0.71±±±±0.03 0.83±±±±0.04 0.94±±±±0.09 1.32±±±±0.02
A15 0.46±±±±0.05 0.65±±±±0.06 0.79±±±±0.07 0.91±±±±0.11 1.15±±±±0.04
A16 0.42±±±±0.07 0.51±±±±0.03 0.67±±±±0.04 0.86±±±±0.08 0.90±±±±0.06
A17 0.47±±±±0.06 0.69±±±±0.06 0.79±±±±0.07 0.92±±±±0.08 1.16±±±±0.09
A18 0.38±±±±0.12 0.54±±±±0.11 0.60±±±±0.08 0.70±±±±0.09 0.85±±±±0.04
A19 0.34±±±±0.09 0.46±±±±0.05 0.57±±±±0.07 0.67±±±±0.04 0.82±±±±0.03
A20 0.48±±±±0.04 0.67±±±±0.03 0.82±±±±0.09 0.93±±±±0.07 1.25±±±±0.08
- 53 -
0
0.2
0.4
0.6
0.8
1
1.2
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on 25% of sugar beet pulp
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on basal liquid medium amended w ith 25% of sugar
beet pulp
Fig. (9): Citric acid production (mg/ml) by different isolates of A. niger
growing on 25% of sugar beet pulp after 12 days incubation.
Fig. (10): Citric acid production (mg/ml) by different isolates of A. niger
growing on basal liquid medium amended with 25% of sugar
beet pulp after 12 days incubation.
- 54 -
Table (9): Citric acid production (mg/ml) by different isolates
of A. niger growing on different concentrations of
molasses after 12 days incubation.
Citric acid production (mg/ml)
Molasses (%) Strain No.
5 10 15 20 25
A1 0.45±±±±0.06 0.60±±±±0.08 0.75±±±±0.07 0.83±±±±0.04 0.94±±±±0.11
A2 0.31±±±±0.05 0.45±±±±0.03 0.52±±±±0.07 0.67±±±±0.08 0.77±±±±0.09
A3 0.35±±±±0.04 0.52±±±±0.09 0.70±±±±0.08 0.74±±±±0.04 0.82±±±±0.03
A4 0.30±±±±0.03 0.49±±±±0.04 0.70±±±±0.05 0.75±±±±0.07 0.84±±±±0.06
A5 0.43±±±±0.08 0.56±±±±0.07 0.71±±±±0.03 0.78±±±±0.03 0.86±±±±0.09
A6 0.27±±±±0.03 0.36±±±±0.05 0.49±±±±0.04 0.57±±±±0.05 0.76±±±±0.10
A7 0.29±±±±0.05 0.41±±±±0.08 0.52±±±±0.05 0.63±±±±0.08 0.77±±±±0.08
A8 0.30±±±±0.06 0.39±±±±0.07 0.59±±±±0.08 0.66±±±±0.06 0.72±±±±0.04
A9 0.38±±±±0.04 0.46±±±±0.14 0.62±±±±0.16 0.71±±±±0.03 0.83±±±±0.03
A10 0.42±±±±0.08 0.49±±±±0.09 0.67±±±±0.03 0.78±±±±0.05 0.87±±±±0.05
A11 0.34±±±±0.06 0.46±±±±0.10 0.53±±±±0.04 0.67±±±±0.03 0.87±±±±0.08
A12 0.39±±±±0.07 0.54±±±±0.07 0.66±±±±0.09 0.77±±±±0.04 0.89±±±±0.07
A13 0.26±±±±0.10 0.37±±±±0.07 0.51±±±±0.05 0.66±±±±0.03 0.72±±±±0.09
A14 0.35±±±±0.12 0.54±±±±0.03 0.65±±±±0.09 0.81±±±±0.07 0.98±±±±0.04
A15 0.33±±±±0.06 0.55±±±±0.07 0.61±±±±0.08 0.79±±±±0.03 0.87±±±±0.10
A16 0.28±±±±0.03 0.35±±±±0.09 0.49±±±±0.06 0.58±±±±0.09 0.67±±±±0.03
A17 0.39±±±±0.09 0.54±±±±0.04 0.69±±±±0.12 0.85±±±±0.10 0.90±±±±0.04
A18 0.24±±±±0.04 0.33±±±±0.07 0.44±±±±0.08 0.56±±±±0.09 0.65±±±±0.65
A19 0.23±±±±0.03 0.31±±±±0.08 0.45±±±±0.07 0.51±±±±0.06 0.59±±±±0.04
A20 0.38±±±±0.09 0.51±±±±0.03 0.64±±±±0.04 0.85±±±±0.10 0.90±±±±0.07
- 55 -
Table (10): Citric acid production (mg/ml) by different
isolates of A. niger growing on basal liquid
medium amended with different concentrations of
molasses after 12 days incubation.
Citric acid production (mg/ml)
Molasses (%) Strain No.
5 10 15 20 25
A1 0.52±±±±0.04 0.69±±±±0.03 0.87±±±±0.06 0.98±±±±0.09 1.17±±±±0.04
A2 0.39±±±±0.03 0.54±±±±0.05 0.63±±±±0.09 0.76±±±±0.07 0.92±±±±0.08
A3 0.43±±±±0.03 0.67±±±±0.09 0.82±±±±0.04 0.90±±±±0.07 0.98±±±±0.06
A4 0.41±±±±0.07 0.65±±±±0.09 0.84±±±±0.06 0.94±±±±0.09 1.04±±±±0.03
A5 0.51±±±±0.10 0.70±±±±0.07 0.87±±±±0.03 0.86±±±±0.04 1.15±±±±0.09
A6 0.30±±±±0.04 0.48±±±±0.03 0.61±±±±0.09 0.77±±±±0.08 0.85±±±±0.07
A7 0.34±±±±0.03 0.50±±±±0.11 0.66±±±±0.08 0.79±±±±0.03 0.87±±±±0.08
A8 0.33±±±±0.07 0.45±±±±0.07 0.63±±±±0.06 0.74±±±±0.12 0.80±±±±0.03
A9 0.45±±±±0.06 0.64±±±±0.05 0.74±±±±0.07 0.90±±±±0.05 1.02±±±±0.11
A10 0.50±±±±0.09 0.68±±±±0.10 0.79±±±±0.03 0.86±±±±0.03 1.09±±±±0.09
A11 0.40±±±±0.11 0.52±±±±0.09 0.64±±±±0.03 0.75±±±±0.13 0.94±±±±0.10
A12 0.43±±±±0.08 0.60±±±±0.08 0.71±±±±0.05 0.87±±±±0.03 0.99±±±±0.05
A13 0.31±±±±0.04 0.42±±±±0.05 0.60±±±±0.03 0.70±±±±0.06 0.78±±±±0.14
A14 0.41±±±±0.05 0.62±±±±0.03 0.80±±±±0.06 0.89±±±±0.16 1.03±±±±0.04
A15 0.40±±±±0.08 0.61±±±±0.13 0.76±±±±0.08 0.88±±±±0.04 0.97±±±±0.05
A16 0.31±±±±0.03 0.44±±±±0.09 0.59±±±±0.08 0.70±±±±0.08 0.78±±±±0.09
A17 0.48±±±±0.03 0.67±±±±0.08 0.78±±±±0.04 0.92±±±±0.09 1.05±±±±0.11
A18 0.28±±±±0.04 0.41±±±±0.07 0.56±±±±0.03 0.65±±±±0.06 0.76±±±±0.07
A19 0.29±±±±0.06 0.38±±±±0.04 0.50±±±±0.05 0.58±±±±0.10 0.65±±±±0.10
A20 0.44±±±±0.05 0.63±±±±0.08 0.78±±±±0.10 0.91±±±±0.05 1.00±±±±0.09
- 56 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on 25% of molasses
0
0.2
0.4
0.6
0.8
1
1.2
Citric acid production (mg/ml)
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
Isolates of A. niger grow ing on basal liquid medium amended w ith
25% of molasses
Fig. (11): Citric acid production (mg/ml) by different isolates of A. niger
growing on 25% of molasses after 12 days incubation.
Fig. (12): Citric acid production (mg/ml) by different isolates of A. niger
growing on basal liquid medium amended with 25% of
molasses after 12 days incubation.
- 57 -
In conclusion from the above results (part I) it can be
noticed that type and concentration of carbohydrate by-products
under the study affect citric acid yield by Aspergillus niger
strains. Increasing carbohydrate by-product concentra-tion led to
increase the production of citric acid, the best concentration for
production was 25% for (maize straw, potato solid wastes
(peels), sugar beet pulp & molasses) (Figs. 13, 14, 15, 16, 17, 18,
19 & 20). Citric acid productivity were obtained by all strains
when using different concentration of the four carbohydrate by-
products when each used alone without any additions after 12
days incubation and the yield enhanced when the fermentation
medium amended with the same concentrations of the mentioned
substrates. Sugar beet pulp and molasses giving the highest yield
by Aspergillus niger strains under the study while maize straw
giving lowest production. The yield on indicator medium and
potato solid wastes almost equal.
It can be concluded that the most potent strains for
production were A1, A4, A5 while A8, A16, A18 & A19 recorded
weak production on indicator medium and the four carbohydrate
by products under the study. Plate (1, 2 & 3) showing production
of citric acid on indicator medium (diameter of clear zone in mm)
of the most potent isolates (A1, A4 & A5), while plate 4, 5, 6 &7
showing clear zone of weak isolates (A8, A16, A18 & A19) (A
particular colony lower the pH of the medium and change the
colour of the indicator medium from purple to yellow).
- 58 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Citric acid production (mg/ml)
A1 A4 A5
Most potent isolates
5%
10%
15%
20%
25%
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
Citric acid production (mg/ml)
A 8 A 16 A 18 A 19
Weak isolates
5%
10%
15%
20%
25%
Fig. (13): Citric acid production (mg/ml) by most potent isolates on basal
medium amended with different concentration of maize straw.
Fig. (14): Citric acid production (mg/ml) by weak isolates on basal medium
amended with different concentration of maize straw.
- 59 -
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/ml)
A1 A4 A5
Most potent isolates
5%
10%
15%
20%
25%
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Citric acid production (mg/ml)
A 8 A 16 A 18 A 19
weak isolates
5%
10%
15%
20%
25%
Fig. (15): Citric acid production (mg/ml) by most potent isolates on basal
medium amended with different concentration of potato solid
wastes.
Fig. (16): Citric acid production (mg/ml) by weak isolates on basal medium
amended with different concentration of potato solid wastes.
- 60 -
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
citric acid production (mg/ml)
A1 A4 A5
Most potent isolates
5%
10%
15%
20%
25%
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/m
l)
A 8 A 16 A 18 A 19
Weak isolates
5%
10%
15%
20%
25%
Fig. (17): Citric acid production (mg/ml) by most potent isolates on basal medium
amended with different concentration of sugar beet pulp.
Fig. (18): Citric acid production (mg/ml) by weak isolates on basal medium
amended with different concentration of sugar beet pulp.
- 61 -
0
0.2
0.4
0.6
0.8
1
1.2
citric acid production (mg/ml)
A1 A4 A5
Most potent isolates
5%
10%
15%
20%
25%
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Citric acid production (mg/ml)
A 8 A 16 A 18 A 19
Weak isolates
5%
10%
15%
20%
25%
Fig. (19): Citric acid production (mg/ml) by most potent isolates on basal
medium amended with different concentration of molasses.
Fig. (20): Citric acid production (mg/ml) by weak isolates on basal medium
amended with different concentration of molasses.
- 62 -
Plate (1): A. niger (I1)
Plate (2): A. niger (I4)
Plate (3): A. niger (I5)
Plates (1,2&3) : Most potent isolates of A. niger for citric acid production
on indicator medium after 8 days incubation.
- 63 -
Plate (6) : A. niger (I18) Plate (7) : A. niger (I19)
Plates (4,5,6&7): Weak isolates of A . niger for citric acid production on
indicator medium after 8 days incubation
- 64 -
- 65 -
1. Influence of uv & γγγγ -rays irradiation on the most potent
strains of A. niger for citric acid production.
The parental selected isolates A1, A4 & A5 were exposed to
uv irradiation at different time intervals: i.e. 2, 5, 10, 15, 20, 25 &
30 minutes beside different doses of γ-rays (25, 50, 100 &
200Gy) as a mutagenic agents in a trail for improving the capacity
for citric acid production.
A. UV irradiation: Data recorded in (table 11) showed number of colony
forming unit of selected isolates of A. niger (A1, A4 & A5) on
indicator medium after exposure to uv irradiation. Increasing time
of exposure to uv irradiation led to decrease number of colony
forming units of the selected isolates of Aspergillus niger (A1, A4
& A5). 100% killing of spores was attended within 25 minutes on
indicator medium for A1 & A5 isolates, while within 20 minutes
for A4. Diameter of Clearing zone (mm) for treated (A1, A4 & A5)
after 2, 4 & 6 days incubation are shown in table (12). Maximum
diameter (88mm & 85mm) obtained by T1 & T2 (5min) for A1,
(84-83mm) by T2 (5min) & T1 (10min) for A4, 85mm obtained by
T4 (2 min) & T2 (5min) for A5, all after 6 days incubation.
Production of parental isolates (wild) A1, A4 & A5 on
indicator medium were: 0.96, 0.95 & 0.99mg/ml respectively
(table 13) after 12 days incubation. When comparing that yield of
citric acid with the obtaining resulting isolates (treated with
- 66 -
Table (11): Number of colonyforming units of selected isolates of A.
niger (A1, A4, & A5) on indicator medium after exposure to
uv irradiation.
Dose (min) A1 A4 A5
0 27 23 30
2 5 4 7
5 5 3 5
10 4 3 4
15 2 1 1
20 1 0 1
25 0 0 0
30 0 0 0
- 67 -
Table (12): Screening of treated isolates obtained after
exposure to uv irradiation of selected A. niger
isolates (A1, A4 and A5) for production of citric
acid on indicator medium in terms of diameters
of clearing zone (mm) at different time intervals.
Diameter of clearing zone (mm)
A1 A4 A5
Incubation period (days)
Time of
exposure
(min)
Type
2 4 6 2 4 6 2 4 6
0 P
(wild) 23 54 71 20 45 63 20 50 68
2 T1 28 62 83 20 45 74 23 58 84 T2 25 58 75 23 59 72 25 50 70 T3 26 60 79 25 58 79 25 62 81 T4 27 60 77 25 58 80 23 62 85 T5 24 58 78 - - - 25 56 75 T6 - - - - - - 22 60 80 T7 - - - - - - 25 60 80 5 T1 30 63 88 20 56 76 20 56 80 T2 28 61 85 25 61 84 25 62 85 T3 27 59 76 23 58 80 27 58 74 T4 24 58 75 - - - 22 56 80 T5 27 60 76 - - - 23 56 80
10 T1 25 61 80 26 60 83 21 55 80 T2 26 59 74 25 58 79 24 63 80 T3 27 61 81 25 58 78 22 55 81 T4 24 60 78 - - - 20 58 80
15 T1 27 61 83 25 60 82 20 53 70 T2 25 59 80 - - - - - -
20 T1 26 63 84 - - - 25 60 65 25 0 - - - - - - - - - 30 0 - - - - - - - - -
No of CFU by A1 = 5 at 2 min.: T1 , T2 , T3 , T4 & T5
- 68 -
uv) at that incubation period, maximum production were obtained
by: A4, T2 (5min), followed by A1T1 (5min) & A4 T1 (10min). The
production were 1.78, 1.73 & 1.70 (mg/ml) respectively as shown
from (table 13 & Figs. 21, 22 & 23). Production of parental
isolates A1, A4 & A5 on basal medium with 25% maize straw
were: 0.73, 0.70 & 0.85 (mg/ml) respectively after 12 days
incubation. Maximum yield from the treated (obtained ones) were
: 1.50, 1.45 (mg/ml) by A4 T2 (5 min) & A4 T1 (10min)
respectively with increase 2 fold (table 14 & Figs. 24, 25 & 26).
Yield of citric acid by parental isolates A1, A4, & A5 on
basal medium with 25% potato solid wastes were: 0.94, 0.91 &
0.95 (mg/ml) respectively after 12 days incubation, while
maximum yield by the treated ones were: 1.85, 1.84 & 1.78
(mg/ml) by A1 T1 (5min), A4 T2 (5min) & A1 T3 (5min)
respectively (table 15, Figs. 27, 28 & 29). On basal medium
amended with 25% sugar beet pulp, the production of parental
isolates A1, A4, & A5 after 12 days incubation were: 1.60, 1.50 &
1.51(mg/ml) respectively after 12 days incubation, while
maximum yield by the treated ones after uv treatment reached to
3.16 & 2.82 (mg/ml) with increase 2 fold by A1 T1 (5min) & A1
T5 (5min) respectively (table 16 & Figs. 30, 31 & 32). As for
basal medium amended with 25% molasses the production after
12 days for the parental isolates A1, A4 & A5 were: 1.22, 1.04 &
1.20 (mg/ml) respectively while the yield ranged from (5.00-
5.62mg/ml) for 17 (seventeen) ones obtained (treated with uv
treatment) from the parental A1 after exposure time 2, 5, 15 & 20
minutes with increase reached to 4.5 folds.
- 69 -
Table (13): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after uv irradiation on
indicator medium. Citric acid production (mg/ml)
A1 A4 A5
Time of exposure
(min) Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 0.59±±±±0.06 0.96±±±±0.05 0.57±±±±0.03 0.95±±±±0.06 0.61±±±±0.07 0.99±±±±0.05
2 T1 1.03±±±±0.08 1.43±±±±0.04 0.93±±±±0.04 1.27±±±±0.03 0.43±±±±0.03 0.65±±±±0.08
T2 0.65±±±±0.09 1.14±±±±0.11 0.85±±±±0.08 1.09±±±±0.08 0.82±±±±0.02 1.02±±±±0.10
T3 0.81±±±±0.05 1.29±±±±0.03 1.06±±±±0.02 1.40±±±±0.06 0.70±±±±0.06 0.91±±±±0.04
T4 0.69±±±±0.03 1.28±±±±0.07 1.10±±±±0.06
*
1.50±±±±0.07 1.15±±±±0.08 1.41±±±±0.03
T5 0.76±±±±0.05 1.36±±±±0.05 0 0 0.86±±±±0.07 1.02±±±±0.06
T6 0 0 0 0 0.76±±±±0.04 0.94±±±±0.03
T7 0 0 0 0 0.92±±±±0.05 1.09±±±±0.07
5 T1
1.50±±±±0.07
*
1.73±±±±0.08 0.99±±±±0.11 1.35±±±±0.07 0.50±±±±0.08 0.72±±±±0.08
T2
1.22±±±±0.06
*
1.54±±±±0.08
1.33±±±±0.04
*
1.78±±±±0.07 1.02±±±±0.11 1.18±±±±0.07
T3 1.17±±±±0.11
*
1.52±±±±0.10
1.08±±±±0.13
*
1.45±±±±0.04 0.93±±±±0.09 1.09±±±±0.08
T4 0.80±±±±0.14 1.24±±±±0.12 0 0 0.78±±±±0.03 0.98±±±±0.06
T5
1.20±±±±0.03
*
1.51±±±±0.08 0 0 1.03±±±±0.06 1.40±±±±0.11
10 T1 0.68±±±±0.04 1.09±±±±0.10
1.27±±±±0.09
*
1.70±±±±0.09 0.52±±±±0.09 0.70±±±±0.09
T2
1.12±±±±0.11
*
1.48±±±±0.03
1.03±±±±0.04 1.41±±±±0.08 0.49±±±±0.07 0.71±±±±0.10
T3
1.09±±±±0.08 1.41±±±±0.07
1.05±±±±0.08 1.37±±±±0.06 0.73±±±±0.04 0.90±±±±0.07
T4 1.10±±±±.0.06
*
1.45±±±±0.08 0 0 0.48±±±±0.02 0.70±±±±0.11
15 T1
1.19±±±±0.06
*
1.54±±±±0.07
1.20±±±±0.13
*
1.56±±±±0.10 0.45±±±±0.05 0.67±±±±0.11
T2 0.71±±±±0.04 1.30±±±±0.09 0 0 0 0
20 T1
1.03±±±±0.05
*
1.59±±±±0.06 0 0 0.069±±±±0.07 1.09±±±±0.06
25 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0
* : increase (1.5-2) fold
- 70 -
0.5
0.60.7
0.80.9
1
1.11.2
1.31.4
1.51.6
1.71.8
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (min)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.80.9
1
1.11.2
1.3
1.4
1.5
1.6
1.71.8
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (min)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
Fig. (21): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after uv irradiation after 12 days incubation on
indicator medium.
Fig. (22): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after uv irradiation after 12 days incubation on
indicator medium.
Fig. (23): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after uv irradiation after 12 days incubation on
indicator medium.
A1T
1 A
1T2
A1T
3 A
1T4
A1T
5
- 71 -
Table (14): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after uv irradiation
growing on basal liquid medium amended with
25% maize straw
Citric acid production (mg/ml)
A1 A4 A5
Time of exposure
(min) Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 0.64±±±±0.06 0.73±±±±0.02 0.61±±±±0.03 0.70±±±±0.06 0.65±±±±0.08 0.85±±±±0.04
2 T1 0.86±±±±0.05 1.05±±±±0.05 0.82±±±±0.08 1.00±±±±0.07 0.70±±±±0.07 0.90±±±±0.07
T2 0.54±±±±0.03 0.90±±±±0.12 0.73±±±±0.04 0.92±±±±0.11 0.63±±±±0.08 0.80±±±±0.09
T3 0.65±±±±0.07 1.00±±±±0.07 0.89±±±±0.06
*
1.11±±±±0.04 0.73±±±±0.04 0.93±±±±0.07
T4 0.61±±±±0.08 0.95±±±±0.11
1.00±±±±0.11
*
1.30±±±±0.08 0.77±±±±0.06 0.95±±±±0.06
T5 0.74±±±±0.09 0.97±±±±0.05 0 0 0.60±±±±0.10 0.78±±±±0.05
T6 0 0 0 0 0.66±±±±0.06 0.88±±±±0.07
T7 0 0 0 0 0.58±±±±0.05 0.79±±±±0.06
5 T1 1.05±±±±0.08
*
1.30±±±±0.06 0.96±±±±0.12
*
1.23±±±±0.05 0.63±±±±0.07 0.85±±±±0.04
T2 0.97±±±±0.08
*
1.18±±±±0.06
1.26±±±±0.03
*
1.50±±±±0.10 0.72±±±±0.03 0.91±±±±0.05
T3 0.94±±±±0.03
*
1.12±±±±0.07
1.02±±±±0.06
*
1.28±±±±0.06 0.70±±±±0.06 0.92±±±±0.03
T4 0.63±±±±0.06 0.82±±±±0.10 0 0 0.67±±±±0.11 0.86±±±±0.09
T5 0.90±±±±0.03 1.09±±±±0.04 0 0 0.61±±±±0.10 0.82±±±±0.06
10 T1 0.70±±±±0.05 0.96±±±±0.07
1.20±±±±0.09
*
1.45±±±±0.04 0.59±±±±0.04 0.78±±±±0.07
T2 0.87±±±±0.09 1.07±±±±0.04 0.90±±±±0.11
*
1.10±±±±0.08 0.82±±±±0.03 0.90±±±±0.10
T3 0.64±±±±0.13 1.00±±±±0.05 0.86±±±±0.03
*
1.08±±±±0.04 0.67±±±±0.07 0.85±±±±0.07
T4 0.91±±±±0.06
*
1.10±±±±0.06 0 0 0.63±±±±0.06 0.82±±±±0.04
15 T1 0.98±±±±0.04
*
1.16±±±±0.10
1.07±±±±0.05 1.31±±±±0.07 0.50±±±±0.04 0.69±±±±0.03
T2 0.82±±±±0.12 1.03±±±±0.04 0 0 0 0
20 T1 0.93±±±±0.05
*
1.22±±±±0.06 0 0 0.45±±±±0.05 0.62±±±±0.07
25 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0
* : increase (1.5-2) fold
- 72 -
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (min)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.8
0.9
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
Fig. (24): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after uv irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
Fig. (25): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after uv irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
Fig. (26): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after uv irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
A1T
1 A
1T2
A1T
3 A
1T4
A1T
5
- 73 -
Table (15): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after uv irradiation
growing on basal liquid medium amended with 25%
potato solid wastes Citric acid production (mg/ml)
A1 A4 A5
Time of exposure
(min) Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 0.71±±±±0.05 0.94±±±±0.05 0.74±±±±0.04 0.91±±±±0.07 0.67±±±±0.03 0.95±±±±0.06
2 T1 0.99±±±±0.02 1.30±±±±0.07 0.98±±±±0.09 1.35±±±±0.04 0.70±±±±0.05 0.98±±±±0.04
T2 0.86±±±±0.04 1.17±±±±0.04 0.89±±±±0.08 1.12±±±±0.07 0.73±±±±0.11 0.93±±±±0.06
T3 0.89±±±±0.03 1.21±±±±0.03 1.04±±±±0.03
*
1.47±±±±0.04 0.72±±±±0.03 1.01±±±±0.03
T4 0.81±±±±0.11 1.10±±±±0.08
1.26±±±±0.05
*
1.73±±±±0.08 0.75±±±±0.09 1.06±±±±0.03
T5 0.90±±±±0.08 1.20±±±±0.06 0 0 0.71±±±±0.04 0.95±±±±0.09
T6 0 0 0 0 0.73±±±±0.07 1.00±±±±0.05
T7 0 0 0 0 0.66±±±±0.06 0.91±±±±0.07
5 T1
1.52±±±±0.06
*
1.85±±±±0.04
1.15±±±±0.06
*
1.47±±±±0.03 0.66±±±±0.03 0.95±±±±0.08
T2
1.35±±±±0.04
*
1.72±±±±0.03
1.47±±±±0.08
*
1.84±±±±0.09 0.72±±±±0.09 1.03±±±±0.04
T3
1.43±±±±0.07
*
1.78±±±±0.06
1.26±±±±0.04
*
1.70±±±±0.10 0.71±±±±0.04 0.96±±±±0.02
T4 0.84±±±±0.09 1.12±±±±0.08 0 0 0.70±±±±0.11 0.93±±±±0.09
T5 1.11±±±±0.06
*
1.45±±±±0.08 0 0 0.68±±±±0.05 0.95±±±±0.04
10 T1 1.03±±±±0.05 1.34±±±±0.03
1.41±±±±0.11
*
1.76±±±±0.05 0.63±±±±0.03 0.85±±±±0.06
T2
1.41±±±±0.07
*
1.75±±±±0.09 1.05±±±±0.09
*
1.45±±±±0.12 0.64±±±±0.07 0.88±±±±0.07
T3 0.97±±±±0.03 1.29±±±±0.11 1.03±±±±0.08
*
1.45±±±±0.07 0.70±±±±0.10 0.94±±±±0.08
T4 1.02±±±±0.09 1.34±±±±0.05 0 0 0.70±±±±0.11 0.93±±±±0.09
15 T1
1.35±±±±0.04
*
1.63±±±±0.12
1.28±±±±0.04
*
1.62±±±±0.08 0.66±±±±0.05 0.92±±±±0.10
T2 0.98±±±±0.03 1.31±±±±0.07 0 0 0 0
20 T1
1.20±±±±0.06
*
1.54±±±±0.04 0 0 0.64±±±±0.06 0.90±±±±0.12
25 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0
* : increase (1.5-2) fold
- 74 -
0.5
0.6
0.70.8
0.9
11.1
1.2
1.3
1.4
1.5
1.61.7
1.8
1.9
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
Fig. (27): Citric acid production (mg/ml) of selected parental isolate A1 and its obtained
isolates after uv irradiation after 12 days incubation on basal medium
amended with 25% potato solid wastes.
Fig. (28): Citric acid production (mg/ml) of selected parental isolate A4 and its obtained
isolates after uv irradiation after 12 days incubation on basal medium
amended with 25% potato solid wastes.
Fig. (29): Citric acid production (mg/ml) of selected parental isolate A5 and its obtained
isolates after uv irradiation after 12 days incubation on basal medium
amended with 25% potato solid wastes
A1T
1 A
1T2
A1T
3 A
1T4
A1T
5
- 75 -
Table (16): Citric acid production (mg/ml) of selected parental
isolates of A. niger (A1, A4 and A5) and obtained isolates
(treated) after uv irradiation growing on basal liquid
medium amended with 25% sugar beet pulp Citric acid production (mg/ml)
A1 A4 A5
Time of exposure
(min) Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild)
1.13±±±±0.03 1.60±±±±0.10 1.13±±±±0.07 1.50±±±±0.09 0.74±±±±0.04 1.51±±±±0.08
2 T1 1.86±±±±0.09 2.16±±±±0.06 1.75±±±±0.04 2.03±±±±0.09 0.66±±±±0.04 1.27±±±±0.04
T2 2.05±±±±0.04 2.32±±±±0.02 1.68±±±±0.08 1.96±±±±0.07 0.64±±±±0.08 1.11±±±±0.07
T3 2.10±±±±0.06 2.36±±±±0.05 1.91±±±±0.06 2.15±±±±0.04 0.78±±±±0.09 1.65±±±±0.03
T4
1.90±±±±0.05 2.18±±±±0.04
2.00±±±±0.05
*
2.30±±±±0.07 0.73±±±±0.05 1.67±±±±0.02
T5 .15±±±±0.05 2.38±±±±0.07 0 0 0.70±±±±0.04 1.47±±±±0.06
T6 0 0 0 0 0.68±±±±0.08 1.37±±±±0.06
T7 0 0 0 0 0.71±±±±0.08 1.49±±±±0.07
5 T1
2.97±±±±0.07
*
3.16±±±±0.04
1.86±±±±0.10 2.23±±±±0.02 0.69±±±±0.09 1.41±±±±0.02
T2
1.96±±±±0.08 2.20±±±±0.06
2.15±±±±0.07
*
2.46±±±±0.06 0.73±±±±0.06 1.51±±±±0.06
T3
2.04±±±±0.02 2.34±±±±0.07
1.98±±±±0.07
*
2.31±±±±0.07 0.65±±±±0.08 1.19±±±±0.07
T4
1.83±±±±0.04 2.12±±±±0.08 0 0 0.72±±±±0.06 1.61±±±±0.05
T5
2.37±±±±0.04
*
2.82±±±±0.05 0 0 0.70±±±±0.05 1.52±±±±0.02
10 T1
2.12±±±±0.07
*
2.50±±±±0.06
2.05±±±±0.09
*
2.42±±±±0.08 0.71±±±±0.06 1.51±±±±0.03
T2
2.17±±±±0.10
*
2.46±±±±0.05
1.88±±±±0.06 2.10±±±±0.06 0.68±±±±0.03 1.39±±±±0.05
T3
2.20±±±±0.09
*
2.52±±±±0.09
1.87±±±±0.10 2.05±±±±0.02 0.70±±±±0.06 1.47±±±±0.11
T4 .79±±±±0.06 2.10±±±±0.12 0 0 0.71±±±±0.04 1.53±±±±0.08
15 T1
1.80±±±±0.10 2.12±±±±0.06
2.00±±±±0.10
*
2.34±±±±0.07 0.69±±±±0.04 1.45±±±±0.04
T2
2.10±±±±0.07
*
2.40±±±±0.09 0 0 00 0
20 T1
2.13±±±±0.08
*
2.58±±±±0.03 0 0 0.67±±±±0.06 1.33±±±±0.07
25 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0
* : increase (1.5-2) fold
- 76 -
25 0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 30 0
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
20 0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 25 30 0
Fig. (30): Citric acid production (mg/ml) of selected parental isolate A1 and its obtained
isolates after uv irradiation after 12 days incubation on basal medium
amended with 25% sugar beet pulp.
Fig. (31): Citric acid production (mg/ml) of selected parental isolate A4 and its obtained
isolates after uv irradiation after 12 days incubation on basal medium
amended with 25% sugar beet pulp.
Fig. (32): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after uv irradiation after 12 days incubation on
basal medium amended with 25% sugar beet pulp.
A1T
1 A
1T2
A1T
3 A
1T4
A1T
5
- 77 -
Maximum yield obtained with A1 T4 (2min), A1 T1 & T3 (5
min), A1 T2 & T4 (10min), the production was: 5.5, 5.6, 5.5, 5.5
and 5.56 respectively, (table 17& Fig. 33). As for A4, 11 (eleven)
obtained ones recorded 2 fold increase in production when
compared to the parental isolate (non-treated) Fig. (34). While A5,
9 (min) derivatives recorded 2 fold increase (table 17 & Fig. 35).
B. γγγγ-rays:
(Table 18) showed number of colony-forming units of
selected isolates of Aspergillus niger (A1, A4 & A5) on indicator
medium after exposure to γ-rays. Decrease of colonies was shown
in (table 19) at doses under the study (25, 50, 100 & 200Gy)
for the three isolates. Data in table (20) revealed increase 2 fold
on indicator medium for the treated ones after γ-rays treatment
after 12 days incubation in derivatives A1: (T1 50Gy; T1-T2 –T3 &
T4 100Gy; T1 & T2200 Gy) (Fig. 36). A4 (Fig. 37) (T1, T2 & T3,
100Gy) A5. (Fig. 38) (T1 & T2 100Gy; T1 & T2 200Gy).
Slight increase in citric acid production in few obtained
ones on based medium with 25% maize straw when compared
with parental isolates (untreated with γ-rays) (table 21 & Figs.
(39, 40 & 41) when the basal medium amended with 25% potato
solid wastes, maximum citric acid production after 12 days
incubation and γ-rays treatment were: 1.80, 1.65, 1.60 & 1.63
(mg/ml) by A1 (T1, T2, T3 & T4, after exposed to 100Gy)
- 78 -
Table (17): Citric acid production (mg/ml) of selected parental isolates
of A. niger (A1, A4 and A5) and obtained isolates (treated) after uv
irradiation growing on basal liquid medium amended with 25%
molasses.
Citric acid production (mg/ml)
A1 A4 A5
Time of exposure
(min) Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 1.12±±±±0.09 1.22±±±±0.10 0.95±±±±0.03 1.04±±±±0.05 1.02±±±±0.09 1.20±±±±0.07
2 T1
4.57±±±±0.07
***
5.38±±±±0.08 2.21±±±±0.10
*
2.62±±±±0.04 1.59±±±±0.04 2.14±±±±0.05
T2
4.40±±±±0.11
***
5.30±±±±0.09 1.70±±±±0.09
*
2.07±±±±0.09 1.79±±±±0.07 2.03±±±±0.04
T3
4.42±±±±0.06
***
5.30±±±±0.12 2.34±±±±0.07
*
2.80±±±±0.02 2.25±±±±0.06
*
3.20±±±±0.03
T4
4.72±±±±0.09
****
5.50±±±±0.04
2.50±±±±0.06
*
2.91±±±±0.05
2.84±±±±0.03
**
3.70±±±±0.03
T5
4.21±±±±0.08
***
5.12±±±±0.06 0 0 2.01±±±±0.04
*
2.72±±±±0.07
T6 0 0 0 0 1.52±±±±0.09
*
2.40±±±±0.08
T7 0 0 0 0 1.72±±±±0.12
*
2.30±±±±0.06
5 T1 4.90±±±±0.04
**** 5.62±±±±0.06 2.30±±±±0.11
* 2.71±±±±0.04 1.52±±±±0.07 2.17±±±±0.06
T2 4.50±±±±0.05
*** 5.36±±±±0.07
2.71±±±±0.08
* 3.05±±±±0.06 1.83±±±±0.05
* 3.12±±±±0.09
T3 4.70±±±±0.07
**** 5.50±±±±0.12
2.49±±±±0.04
* 2.90±±±±0.07 0.97±±±±0.03 1.58±±±±0.10
T4 4.16±±±±0.08
*** 5.02±±±±0.04 0 0 1.62±±±±0.13 2.35±±±±0.07
T5 4.23±±±±0.12
*** 5.12±±±±0.09 0 0 2.14±±±±0.07
* 2.40±±±±0.04
10 T1 4.66±±±±0.10
*** 5.44±±±±0.03
2.60±±±±0.03
* 2.98±±±±0.07 1.97±±±±0.07
* 2.68±±±±0.06
T2 4.44±±±±0.06
**** 5.50±±±±0.08 2.30±±±±0.13
* 2.78±±±±0.09 1.70±±±±0.08
1.98±±±±0.08
T3 4.30±±±±0.07
** 5.15±±±±0.13 2.27±±±±0.06
* 2.70±±±±0.05 1.91±±±±0.08
* 2.52±±±±0.09
T4
4.81±±±±0.05
****
5.56±±±±0.07 0 0 1.60±±±±0.07 2.17±±±±0.05
15 T1 4.59±±±±0.09
*** 5.39±±±±0.10
2.50±±±±0.08
* 2.84±±±±0.07 1.79±±±±0.04 2.30±±±±0.03
T2 4.48±±±±0.03
*** 5.32±±±±0.05 0 0 0 0
20 T1 4.45±±±±0.06
*** 5.34±±±±0.05 0 0 1.66±±±±0.03 2.21±±±±0.09
25 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0
****:increase (4.5fold)***:increase(4fold)**:increase (3fold)*increase (2 fold)
- 79 -
0.1
0.50.9
1.31.7
2.12.5
2.93.3
3.74.1
4.54.9
5.35.7
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 0
2 5 10 15 20 25 30 0
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (m in)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 0
2 5 10 15 20 25 30 0
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
3.3
3.7
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (min)
p 0T1 T2T3 T4T5 T6T7 0T1 T2T3 T4T5 0T1 T2T3 T40 T1T2 0T1 00 00
2 5 10 15 20 25 30 0
Fig. (33): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
Fig. (34): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
Fig. (35): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after uv irradiation after 12 days incubation on
basal medium amended with 25% molasses
A1T
1 A
1T2
A1T
3 A
1T4
A1T
5
- 80 -
Table (18): Number of colony-forming units of selected
isolates of A. niger (A1, A4 and A5) on indicator
medium after exposure to gamma rays.
Dose (Gy) A1 A4 A5
0
25
50
100
200
27
1
3
4
2
23
1
2
3
1
30
1
1
2
2
- 81 -
Table (19): Screening of treated isolates obtained after gamma rays
irradiation of selected A. niger isolates (A1, A4 and A5) for
production of citric acid and obtained isolates on indicator
medium in terms of diameter of clearing zone (mm) at
different time intervals.
Diameter of clearing zone (mm)
A1 A4 A5
Days days days Dose
(Gy) Type 2 4 6 2 4 6 2 4 6
0 P
(wild) 23 54 71 20 45 63 20 50 68
25 T1 24 55 69 22 50 64 21 54 68
50 T1 25 57 72 23 51 67 22 55 70
T2 21 57 71 24 53 69 0 0 0
T3 21 50 70 0 0 0 0 0 0
100 T1 24 56 74 23 54 70 25 58 73
T2 24 57 72 24 55 71 24 57 73
T3 22 56 70 22 53 70 0 0 0
T4 23 56 71 0 0 0 0 0 0
200 T1 23 55 72 23 51 65 22 54 70
T2 22 56 74 0 0 0 21 53 72
- 82 -
Table (20): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after gamma rays
irradiation on indicator liquid medium Citric acid production (mg/ml)
A1 A4 A5 Dose (Gy.)
Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P
(wild) 0.59±±±±0.09 0.96±±±±0.10 0.57±±±±0.08 0.95±±±±0.04 0.61±±±±0.05 0.99±±±±0.07
25 T1 0.55±±±±0.06 1.40±±±±0.011 0.60±±±±0.06 1.40±±±±0.05 0.63±±±±0.08 1.80±±±±0.04
50 T1 1.00±±±±0.07
*
1.90±±±±0.06 0.63±±±±0.09 1.76±±±±0.04
1.23±±±±0.06 1.87±±±±0.09
T2 0.79±±±±0.09 1.80±±±±0.05 0.72±±±±0.03 1.69±±±±0.08 0 0
T3 0.74±±±±0.05 1.74±±±±0.07 0 0 0 0
100 T1
1.58±±±±0.11
*
2.15±±±±0.04
1.12±±±±0.12
*
1.82±±±±0.09
1.54±±±±0.09
*
2.10±±±±0.03
T2
1.44±±±±0.05
*
2.12±±±±0.12
1.22±±±±0.04
*
1.87±±±±0.06
1.59±±±±0.04
*
2.04±±±±0.04
T3
1.20±±±±0.07
*
2.00±±±±0.08
1.14±±±±0.07
1.79±±±±0.06 0 0
T4
1.26±±±±0.08
*
1.96±±±±0.03 0 0 0 0
200 T1 1.39±±±±0.06
*
2.10±±±±0.07 0.98±±±±0.06 1.40±±±±0.03
1.25±±±±0.11 1.95±±±±0.10
T2
1.49±±±±0.03
*
2.15±±±±0.08 0 0
1.39±±±±0.08
*
1.98±±±±0.06
* : increase (2 fold)
- 83 -
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.81.92
2.1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
Fig. (36): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on indicator liquid medium
Fig. (37): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on indicator liquid medium
Fig. (38): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on indicator liquid medium
- 84 -
Table (21): Citric acids production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after gamma rays
irradiation growing on basal liquid medium
amended with 25% maize straw
Citric acid production (mg/ml)
A1 A4 A5 Dose (Gy.)
Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 0.64±±±±0.11 0.73±±±±0.09 0.61±±±±0.10 0.70±±±±0.13 0.65±±±±0.06 0.85±±±±0.03
25 T1 0.63±±±±0.08 0.70±±±±0.06 0.67±±±±0.05 0.71±±±±0.09 0.64±±±±0.05 0.76±±±±0.09
50 T1 0.75±±±±0.06 0.83±±±±0.04 0.70±±±±0.09 0.75±±±±0.04 0.70±±±±0.09 0.84±±±±0.08
T2 0.72±±±±0.05 0.88±±±±0.11 0.69±±±±0.07 0.77±±±±0.05 0 0
T3 0.70±±±±0.07 0.79±±±±0.10 0 0 0 0
100 T1 0.94±±±±0.03 1.10±±±±0.03 0.70±±±±0.05 0.80±±±±0.06 0.90±±±±0.11 1.03±±±±0.05
T2 0.78±±±±0.06 1.00±±±±0.06 0.76±±±±0.08 0.84±±±±0.07 0.95±±±±0.04 1.09±±±±0.07
T3 0.70±±±±0.02 0.94±±±±0.07 0.71±±±±0.04 0.79±±±±0.08 0 0
T4 0.76±±±±0.10 0.96±±±±0.09 0 0 0 0
200 T1 0.80±±±±0.09 1.02±±±±0.13 0.68±±±±0.03 0.75±±±±0.06 0.72±±±±0.06 0.89±±±±0.12
T2 0.90±±±±0.08 1.05±±±±0.06 0 0 0.80±±±±0.07 0.94±±±±0.03
- 85 -
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
Fig. (39): Citric acid production (mg/ml) of selected parental isolate A1 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
Fig. (40): Citric acid production (mg/ml) of selected parental isolate A4 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
Fig. (41): Citric acid production (mg/ml) of selected parental isolate A5 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% maize straw.
- 86 -
with increase 1.5 fold (table 22) (Fig. 42), no increase in
production obtained in the derivatives for A4 & A5 after all
doses, of γ-rays under the study (Figs. 43, 44). As for sugar beet
pulp, slight increase in citric acid production for some obtained
ones on that carbohydrate by-product (table 23, Figs 45, 46 & 47)
when compared to the parental isolates (untreated with γ-rays).
On basal medium amended with 25% molasses, increase in
production for ten (10) obtained ones for A1 after treatment with
γ- rays. 2 fold recorded after 12 days incubation by A1 (T1 100Gy)
& 1.5 fold for (T1) 25Gy; (T1, T2, T3) 50Gy; (T2, T3, T4) 100Gy &
(T1, T2) 200Gy (tables 24 & 48). Also increases in production
reached to 1.5 folds recorded in the table for some obtained ones
in this carbohydrate by-product by A4 & A5 strains (Figs. 49 &
50).
From the data obtained in part II it can be concluded that uv
treatment affect positively citric acid production by selected
isolates of A. niger growing on different carbohydrate by
products. Molasses was the best carbohydrate by product for citric
acid production when added at 25% conc. to the basal medium
after 12 days incubation (increase reached to 5 fold in some
obtained ones after uv treatment from the parental isolate, A1 as
mentioned followed by sugar beet pulp, while the lowest yield
recorded with maize straw. Production by treated isolates (after
uv treatment) growing on indicator medium & basal medium with
potato solid wastes almost equal, both better than with maize
straw but lowest than molasses and sugar beet pulp. As for
γ-rays, increase in
- 87 -
Table (22): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after gamma rays
irradiation growing on basal liquid medium
amended with 25% potato solid wastes.
Citric acid production (mg/ml)
A1 A4 A5 Dose (Gy.)
Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P (wild) 0.71±±±±0.08 0.94±±±±0.06 0.74±±±±0.03 0.91±±±±0.08 0.67±±±±0.04 0.95±±±±0.03
25 T1 0.68±±±±0.07 0.90±±±±0.04 0.71±±±±0.08 0.94±±±±0.09 0.70±±±±0.06 0.97±±±±0.10
50 T1 0.89±±±±0.10 0.96±±±±0.06 0.74±±±±0.07 0.96±±±±0.04 0.77±±±±0.05 1.00±±±±0.03
T2 0.76±±±±0.03 1.03±±±±0.05 0.76±±±±0.05 0.98±±±±0.06 0 0
T3 0.74±±±±0.07 1.00±±±±0.06 0 0 0 0
100 T1 1.00±±±±0.09
*
1.80±±±±0.07 0.80±±±±0.04 1.10±±±±0.10 0.93±±±±0.09 1.14±±±±0.12
T2 0.86±±±±0.06
*
1.65±±±±0.08 0.82±±±±0.06 1.08±±±±0.09 1.00±±±±0.03 1.20±±±±0.10
T3 0.76±±±±0.08
*
1.60±±±±0.09 0.79±±±±0.04 1.05±±±±0.06 0 0
T4 0.80±±±±0.06
*
1.63±±±±0.04 0 0 0 0
200 T1 0.85±±±±0.12 1.15±±±±0.03 0.76±±±±0.04 1.00±±±±0.07 0.80±±±±0.04 0.99±±±±0.07
T2 0.96±±±±0.10 1.20±±±±0.09 0 0 0.84±±±±0.06 1.05±±±±0.08
* : increase (1.5 fold)
- 88 -
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Citric acid pro
duction (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.8
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
Fig. (42): Citric acid production (mg/ml) of selected parental isolate A1 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% potato solid wastes.
Fig. (43): Citric acid production (mg/ml) of selected parental isolate A4 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% potato solid wastes.
Fig. (44): Citric acid production (mg/ml) of selected parental isolate A5 and its obtained
isolates after gamma rays irradiation after 12 days incubation on basal
medium amended with 25% potato solid wastes.
- 89 -
Table (23): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after gamma rays
irradiation growing on basal liquid medium
amended with 25% sugar beet pulp.
Citric acid production (mg/ml)
A1 A4 A5 Dose (Gy.)
Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P
(wild) 1.13±±±±0.07 1.60±±±±0.06 1.13±±±±0.09 1.50±±±±0.04 0.74±±±±0.09 1.51±±±±0.13
25 T1 0.94±±±±0.11 1.20±±±±0.10 0.72±±±±0.06 1.00±±±±0.07 0.76±±±±0.05 1.15±±±±0.06
50 T1 1.20±±±±0.10 1.56±±±±0.13 0.80±±±±0.07 1.07±±±±0.08 1.13±±±±0.04 1.60±±±±0.07
T2 1.16±±±±0.08 1.67±±±±0.04 1.09±±±±0.04 1.30±±±±0.12 0 0
T3 1.00±±±±0.07 1.55±±±±0.06 0 0 0 0
100 T1 1.60±±±±0.06 2.10±±±±0.09 1.00±±±±0.03 1.76±±±±0.14 1.46±±±±0.08 1.94±±±±0.09
T2 1.34±±±±0.03 1.87±±±±0.06 1.04±±±±0.08 1.70±±±±0.09 1.42±±±±0.07 1.81±±±±0.03
T3 1.11±±±±0.07 1.67±±±±0.07 1.00±±±±0.07 1.65±±±±0.06 0 0
T4 1.25±±±±0.06 1.73±±±±0.04 0 0 0 0
200 T1 1.40±±±±0.08 1.85±±±±0.02 0.95±±±±0.06 1.35±±±±0.04 1.00±±±±0.05 1.60±±±±0.04
T2 1.46±±±±0.05 1.99±±±±0.07 0 0 1.17±±±±0.04 1.67±±±±0.08
- 90 -
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.81.92
2.1
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.8
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.81.9
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
Fig. (45): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on basal medium amended with 25% sugar beet pulp.
Fig. (46): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on basal medium amended with 25% sugar beet pulp.
Fig. (47): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after gamma rays irradiation after 12 days
incubation on basal medium amended with 25% sugar beet pulp.
- 91 -
Table (24): Citric acid production (mg/ml) of selected
parental isolates of A. niger (A1, A4 and A5) and
obtained isolates (treated) after gamma rays
irradiation growing on basal liquid medium
amended with 25% molasses.
Citric acid production (mg/ml)
A1 A4 A5 Dose (Gy.)
Type
6 days 12 days 6 days 12 days 6 days 12 days
0 P
(wild) 1.09±±±±0.04 1.20±±±±0.07 0.92±±±±0.09 1.00±±±±0.08 1.00±±±±0.06 1.17±±±±0.10
25 T1 1.00±±±±0.08
*
1.88±±±±0.06 0.95±±±±0.03
*
1.5±±±±0.10 0.98±±±±0.04
*
2.03±±±±0.08
50 T1 1.20±±±±0.06
*
2.21±±±±0.05 0.99±±±±0.04
*
1.87±±±±0.05 1.25±±±±0.06
*
2.07±±±±0.05
T2 1.12±±±±0.04
*
2.08±±±±0.06 1.01±±±±0.09
*
1.94±±±±0.03 0 0
T3 1.03±±±±0.03
*
1.95±±±±0.04 0 0 0 0
100 T1 1.60±±±±0.09
**
2.40±±±±0.03 1.08±±±±0.03 1.75±±±±0.07
1.65±±±±0.03
*
2.12±±±±0.07
T2 1.44±±±±0.8
*
2.25±±±±0.06 1.12±±±±0.05
*
1.72±±±±0.04
1.61±±±±0.08
*
2.10±±±±0.04
T3 1.26±±±±0.07
*
2.15±±±±0.07 1.10±±±±0.07
*
1.80±±±±0.06 0 0
T4 1.34±±±±0.04
*
2.25±±±±0.09 0 0 0 0
200 T1 1.43±±±±0.03
*
2.15±±±±0.10 0.97±±±±0.06
*
1.70±±±±0.03 1.07±±±±0.09
*
1.97±±±±0.03
T2 1.48±±±±0.09
*
2.20±±±±0.08 0 0 1.20±±±±0.10
*
2.08±±±±0.09
* : increase (1.5 fold) * * : increase (2 fold)
- 92 -
0.10.30.50.70.91.11.31.51.71.92.12.32.5
Citric acid
production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T225 50 100 200 0
0.1
0.3
0.5
0.7
0.9
1.1
1.3
1.5
1.7
1.9
2.1
2.3
2.5
Citric acid production (mg/ml)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
0.10.20.30.40.50.60.70.80.91
1.11.21.31.41.51.61.71.81.92
2.1
Citric acid production (mg/m
l)
Dose of gamma rays (Gy)
p 0
T1 0
T1 T2
T3 0
T1 T2
T3 T4
0 T1
T2
25 50 100 200 0
Fig. (48): Citric acid production (mg/ml) of selected parental isolate A1 and its
obtained isolates after gamma rays irradiation after 12 days incubation on
basal medium amended with 25% molasses.
Fig. (49): Citric acid production (mg/ml) of selected parental isolate A4 and its
obtained isolates after gamma rays irradiation after 12 days incubation on
basal medium amended with 25% molasses.
Fig. (50): Citric acid production (mg/ml) of selected parental isolate A5 and its
obtained isolates after gamma rays irradiation after 12 days incubation on
basal medium amended with 25% molasses.
- 93 -
production (2 fold) with some obtained ones on molasses only.
UV treatment was more effective than γ-rays in producing
number of obtained isolates of A. niger yielding more production
of citric acid than the parental strains.
2. Influence of uv irradiation on weak isolates of A. niger for
citric acid production:
Table (25) showed number of colony forming units of weak
isolates of A. niger (A8, A16, A18, & A19) on indicator medium
after uv treatment. Decrease of colony forming units were
obtained after 10 & 15 minutes from exposure. 100% killing of
spores after 20 minutes of exposure for A16 while within 25
minutes for A8, A18 & A19.
Production of these parental isolates after 12 days
incubation on that medium were: 0.74, 0.70, 0.69 & 0.60 (mg/ml)
respectively. Interestingly, increase of production fold reached to
5, 6, 7, 8 & 9 folds as recorded in (table 27) when comparing the
production with the wild (parental) isolates after uv irradiation.
Maximum production by A8 were obtained by T1, T2, T3, T4, & T5
(10 minutes), the production were 3.6, 3.5, 3.43, 3.04 & 3.15
(mg/ml) respectively (Fig. 51) with increase of 4.5 folds. Also 10
minutes achieved maximum production for A16, obtained by T1,
T2, T3, T4 & T5 as shown from the table (27) and Fig.(52), the
production after 12 days were: 5.2, 5.15, 4.2, 4.87 & 4.13 (mg/ml)
- 94 -
respectively with increase 6 & 7 folds. Again 10 minutes achieved
maximum production for A18 by T1, T2, T3, T4 & T5 also T1
(15min), the production were: 5.2, 5.17, 4.65, 4.8, 4.0 & 4.2
(mg/ml) respectively with 6-7 fold increase (Fig. 53). Production
reached to 8.5 fold increase with A19, T1 & T2 (10 minutes),
production were: 5.35 & 5.25 (mg/ml) respectively (Fig. 54).
In conclusion, pronounced increase in citric acid production
recorded in this study by weak isolates when exposed to U.V
treatment growing on indicator medium, when compared the
derivatives ones with the parental strains. A19, T1 (10 minutes)
was chosen for PCR studies, 9 fold increase was obtained after
12 days incubation on indicator medium as compared to the
parental isolate. Table (28) showed citric acid production of this
isolate on different carbohydrate by-products in 12 days
incubation. Molasses recorded the highest yield for this obtained
isolate followed by sugar beet pulp and potato solid wastes, maize
straw recorded the lowest the lowest production (Fig. 55 & 56).
Plate 7- showed citric acid production of parental isolate A19
(clear zone) on indicator medium after 12 days incubation while
plate 8 showed derivative isolate of A19 after uv treatment
(10mim) after 6 days incubation (A19 , T1).
- 95 -
Table (25): Number of colony-forming units of weak isolates
of A. niger (I8, I16, I18 and I19) on indicator
medium after uv irradiation.
Dose
(min)
A8 A16 A18 A19
0
2
5
10
15
20
25
30
13
11
10
5
3
1
0
0
11
10
10
4
1
0
0
0
12
10
9
4
2
1
0
0
10
8
8
3
1
1
0
0
- 96 -
Table (26): Screening of treated isolates obtained after uv
irradiation of parental weak isolates of A. niger
(A8, A16, A18 and A19) for production of citric acid
on indicator medium in terms of diameter of
clearing zone (mm) at different time intervals.
Diameter of clearing zone (mm)
A8 A16 A18 A19
days days days days
Dose (min)
Type
2 4 6 2 4 6 2 4 6 2 4 6
0 P
(Wild) 20 30 34 20 22 25 20 25 25 10 17 20
2 T1 21 32 36 21 25 29 22 26 30 15 20 23
5 T1 22 34 38 21 27 30 21 29 32 19 22 25
10 T1 25 60 85 24 62 87 24 64 88 28 62 90
T2 27 56 75 28 64 87 23 61 87 25 60 90
T3 25 58 75 25 60 79 22 55 80 27 58 82
T4 23 51 70 24 64 84 22 58 83 24 61 81
T5 23 54 72 24 53 76 21 51 75 25 60 80
15 T1 22 50 58 22 50 75 21 54 77 23 67 83
T2 20 32 50 21 47 68 22 50 70 22 54 72
T3 21 30 45 22 49 77 21 48 71 22 57 75
20 T1 20 29 38 0 0 0 21 45 60 21 51 70
25 0 0 0 0 0 0 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0 0 0 0 0 0 0
- 97 -
Table (27): Citric acid production by parental weak isolates
(A8, A16, A18 and A19) of A. niger and obtained isolates after uv
irradionlion on indicator medium at different time intervals.
***** : increase (8.5 fold) **** : increase (8 fold) *** : increase (7 fold) ** : increase (6 fold) * : increase (5 fold)
Citric acid production (mg/ml)
A8 A16 A18 A19
Days days days days
Time of exposure
to u.v. (min)
Type
6 12 6 12 6 12 6 12
0 P
(Wild) 0.41±±±±0.05 0.74±±±±0.09 0.35±±±±0.04 0.70±±±±0.08 0.38±±±±0.07 0.69±±±±0.03 0.28±±±±0.04 0.60±±±±0.08
2 T1 0.53±±±±0.07 0.80±±±±0.09 0.50±±±±0.08 0.73±±±±0.04 0.52±±±±0.07 0.75±±±±0.06 0.40±±±±0.07 0.83±±±±0.04
5 T1 0.74±±±±0.04 0.83±±±±0.09 0.61±±±±0.07 0.80±±±±0.03 0.65±±±±0.04 0.90±±±±0.07 0.42±±±±0.07 0.90±±±±0.03
10 T1
2.70±±±±0.05
*
3.60±±±±0.08
2.57±±±±0.06
***
5.20±±±±0.05
0.65±±±±0.03
***
5.20±±±±0.04
2.70±±±±0.04
*****
5.35±±±±0.07
T2
2.40±±±±0.08
3.50±±±±0.06
2.51±±±±0.05
***
5.15±±±±0.06
2.60±±±±0.07
***
5.17±±±±0.06
2.65±±±±0.05
*****
5.25±±±±0.05
T3
2.32±±±±0.09
3.43±±±±0.05
2.25±±±±0.06
**
4.20±±±±0.07
2.28±±±±0.07
**
4.65±±±±0.03
2.20±±±±0.06
***
4.80±±±±0.04
T4
1.85±±±±0.03 3.04±±±±0.04
2.38±±±±0.04
***
4.87±±±±0.04
2.40±±±±0.08
***
4.80±±±±0.07
2.15±±±±0.07
***
4.60±±±±0.03
T5
1.93±±±±0.06 3.15±±±±0.08
2.12±±±±0.09
**
4.13±±±±0.08
2.17±±±±0.06
*
4.00±±±±0.08
2.12±±±±0.08
***
4.45±±±±0.07
15 T1 1.70±±±±0.07 2.50±±±±0.09
2.10±±±±0.09
**
3.90±±±±0.09
2.20±±±±0.05
**
4.12±±±±0.06
2.29±±±±0.11
****
4.78±±±±0.06
T2 1.60±±±±0.04 2.03±±±±0.04
1.86±±±±0.08
*
3.41±±±±0.03
1.92±±±±0.11
*
3.50±±±±0.07
2.02±±±±0.14
**
3.65±±±±0.09
T3 1.52±±±±0.04 1.95±±±±0.10
1.93±±±±0.07
*
3.52±±±±0.06
1.99±±±±0.10
3.23±±±±0.06
2.09±±±±0.07
**
3.86±±±±0.04
20 T1 1.44±±±±0.03 1.75±±±±0.05 0 0
1.88±±±±0.02 3.00±±±±0.05
1.98±±±±0.05
**
3.50±±±±0.06
25 0 0 0 0 0 0 0 0 0
30 0 0 0 0 0 0 0 0 0
- 98 -
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
3.3
3.7
Citric acid production (mg/m
l)
Time of exposure to u.v light irradiation (m in)
p 0
T1 0
T1 0
T1 T2
T3 T4
T5 0
T1 T2
T3 0
T1 0
0 0
0
2 5 10 15 20 25 30 0
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
3.3
3.7
4.1
4.5
4.9
5.3
Citric acid production (mg/ml)
Time of exposure to u.v light irradiation (min)
p 0
T1 0
T1 0
T1 T2
T3 T4
T5 0
T1 T2
T3 0
T1 0
0 0
0
2 5 10 15 20 25 30 0
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
3.3
3.7
4.1
4.5
4.9
5.3
Citric acid production (mg/m
l)
Time of exposure to u.v light irradiation (m in)
p 0
T1 0
T1 0
T1 T2
T3 T4
T5 0
T1 T2
T3 0
T1 0
0 0
0
2 5 10 15 20 25 30 0
Fig. (51): Citric acid production (mg/ml) of parental weak isolate A8 and its
obtained isolates after exposure to u.v. irradiation after 12 days
incubation on indicator medium
Fig. (52): Citric acid production (mg/ml) of parental weak isolate A16 and its
obtained isolates after exposure to u.v. irradiation after 12 days
incubation on indicator medium
Fig. (53): Citric acid production (mg/ml) of parental weak isolate A18 and its
obtained isolates after exposure to u.v. irradiation after 12 days
incubation on indicator medium
- 99 -
0.1
0.5
0.9
1.3
1.7
2.1
2.5
2.9
3.3
3.7
4.1
4.5
4.9
5.3
Citric acid production (mg/m
l)
Time of exposure to u.v light irradiation (m in)
p 0
T1 0
T1 0
T1 T2
T3 T4
T5 0
T1 T2
T3 0
T1 0
0 0
0
2 5 10 15 20 25 30 0
0
1
2
3
4
5
6
7
Citric acid production (mg/ml)
Maize straw Potato solid wastes Sugar beet pulp Molasses
Carbohydrarte by products
0
5
10
15
20
25
30
35
40
45
Substrate consumed (%)
Maize straw Potato solid wastes Sugar beet pulp Molasses
Carbohydrarte by products
Fig. (54): Citric acid production (mg/ml) of parental weak isolate A19 and its obtained
isolates after uv irradiation after 12 days incubation on indicator medium
.
Fig. (55): Citric acid production (mg/ml) by A19 (T1 u.v.10) of A. niger on different
carbohydrate by products after 12 days incubation.
Fig. (56): Substrate consumed (%) by A19 (T1 u.v.10) of A. niger on different
carbohydrate by products after 12 days incubation.
- 100 -
Table (28): Citric acid production (mg/ml) by A19 (T1 u.v.10)
of A. niger on different carbohydrate by
products after 12 days incubation.
Carbohydrate
by products Citric acid mg/ml Substrate consumed (%)
Maize straw 2.05 0.6
Potato solid wastes 2.83 2.5
Sugar beet pulp 3.80 15
Molasses 6.10 41.06
Weight of substrate before fermentation - Weight of substrate after fermentation Substrate
consumed % =
Weight of substrate before fermentation X 100
- 101 -
Plate (7): Wild (Parental isolate) of A.niger (I 19) on indicator medium
after 8 days incubation.
(Obtained isolate)
Plate (8): Derivative isolate of A. niger (I19) after exposure to U.V. light
irradiation (10 minutes) after 6 days incubation on indicator
medium (A19 T1).
- 102 -
- 103 -
From the previous study and the obtained results,
Aspergillus niger A1 (potent strain) and A1T1 (5 min) in addition
to A19 (weak strain) and A19 T1 (10 min) were chosen for PCR and
protein electrophoresis studies.
RAPD analysis by using PCR has been used successfully to
indentify genetic variability on the molecular level between wild
strain of Aspergillus niger (A1 & A19) and its treated isolates with
uv irradiation at 5 min &10 min respectively i.e. (A1 T1) & (A19
T1). Ten arbitrary primers were used for PCR amplification of
total DNAs of the (A1&A1T15min). Figures (57-61) showed the
amplified product of bands, and total number of the amplified
RAPD bands produced by the 10 arbitrary primers that is present
in table (29).
As shown from the mentioned table and figures, RAPD
analysis revealed that three primers (OPB-10, OPB-08 & OPA-
15) showed no polymorphism among two strains (A1 and A1T1).
On the other hand, it was found that seven primers (OPO-14,
OPO-02, OPA-05, OPA-04, OPA-18, OPC-14 & OPB-05)
showed variations in number of bands between the two mentioned
strains. These variations come from the mutagenic effect of uv
irradiation on the Aspergillus niger .
- 104 -
Table (29): Number of amplified DNA bands scored for the
wild strain A. niger A1 (potent isolate) and its obtained isolate
(A1 T1 5min) by uv irradiation.
No. Primers
Wild
strain
(A1)
Obtained
isolate (A1T1
5min)
No of
total
bands
No of
shared
bands
1
2
3
4
5
6
7
8
9
10
OPO-14
OPO-02
OPB-10
OPB-08
OPA-05
OPA-04
OPA-15
OPA-18
OPC-14
OPB-05
4
4
2
4
10
1
4
3
2
5
3
2
2
4
8
4
4
4
1
3
7
6
4
8
18
5
8
7
3
8
2
2
2
4
8
1
4
3
1
3
Data in table (30) indicated the unique positive and/or
negative marker and their molecular weight (size) generated by
RAPD analysis for the two strains (A1& A1,T1 5 min). Data
revealed that five positive unique markers identified the
mutantA1T1 5min, amplified by OPO-14 primer at molecular
weight of 4245 bp (base pairs) OPA-18 primer, at molecular
weight of 811bp, and OPA-04 primer at molecular weight 572,
219 and 135bp, these five bands found only on A1T1.
- 105 -
In the wild isolate A1 nine unique markers amplified by
OPO-02 primer at molecular weight of 2115 and 811bp, OPO-14
primer at molecular weight of 573 & 325bp, OPC-14 primer at
molecular weight of 548bp, OPA-05 primer at molecular weight
604 & 480 bp and OPB-05 primer at molecular weight 453 &
272bp.
Table (30): Positive and negative unique RAPD markers and
their molecular weight of the two strains of A.
niger (A1 & A1 T1 5 min).
Primers Approx band size in bp A1 A1 T1
OPO-02 2115
811
1
1
0
0
OPO-14
4245
573
325
0
1
1
1
0
0
OPC-14 548 1 0
OPA-05 604
480
1
1
0
0
OPB-05 453
272
1
1
0
0
OPA-18 811 0 1
OPA-04
572
219
135
0
0
0
1
1
1 Presence of a band in a genotype = 1 Absence of the shared band in the other genotype = 0
- 106 -
Fig. (57): RAPD banding patterns amplified for A. niger (A1) and its
obtained isolate(A1T15min) using primers(OPO-14&OPO-02)
M (DNA marker) = 5Kb ladder.
T1 A1 M T1 A1
OPB-10 OPB-08
Fig. (58): RAPD banding patterns amplified for A. niger (A1) and its
obtained isolate (A1T15min) using primers(OPB-10&OPB-08)
M (DNA marker) = 1Kb ladder.
- 107 -
A1 T1 M
OPA-05
Fig (59): RAPD banding patterns amplified for A. niger (A1) and its
obtained isolate (A1 T1 5min) using primer (OPA-05) M
(DNA marker) = 1 Kb ladder.
A1 T1 M A1 T1 M A1 T1
OPA-18 OPA-15 OPA-04
Fig. (60): RAPD banding patterns amplified for A. niger (A1) and its
obtained isolate (A1T1 5min) using primers (OPA-04, OPA-15
& OPA-18) M (DNA marker) = 1 Kb ladder.
- 108 -
A1 T1 M A1 T1
OPB-05 OPC-14
Fig. (61): RAPD banding patterns amplified for A. niger (A1) & its
obtained isolate (A1 T1 5min) using primers (OPC-14 & OPB-
05) M (DNA marker) = 1kb ladder.
Figures (62-71) showed the amplified products of bands and total number of the amplified RAPD bands produced by the 10 random primers for A. niger wild strain A19 (weak isolate) & its obtained isolate after uv irradiation (A19T1 10min) and presented in table (31). RAPD analysis revealed that 3 out of 10 primers OPC-20, OPO-04 & OPB-05 showed similarities in bands between the two strain (wild & obtained isolate). On the other hand, it was found that the amplified product by other seven primers (OPG-05, OPB-15, OPB-06, OPA-20, OPH-15, OPC-10 & OPB-07) showed difference in number of bands between the genotypes A19 & A19 T1 (10min). These variations come from the mutagenic effect of uv irradiation on A. niger.
- 109 -
Table (31): Number of amplified DNA bands scored for the
wild strain of A. niger A19 and its obtained isolate
(A19T1 10 min) by uv irradiation.
No. Primers Wild strain
(A19)
Obtained
isolate (A19T1
10min)
No of
total
bands
No of
shared
bands
1
2
3
4
5
6
7
8
9
10
OPG-05
OPH-15
OPC-10
OPC-20
OPA-20
OPO-04
OPB-15
OPB-05
OPB-07
OPB-06
2
5
7
8
4
6
4
6
5
4
3
8
6
8
6
6
3
6
6
3
5
13
13
16
10
12
7
12
11
7
2
1
6
8
4
6
3
6
0
3
- 110 -
M A19 T1
OPG-05
Fig. (62): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolate (A19 T1 10 min) using primer OPG-05 M
(DNA marker) = 1kb ladder.
- 111 -
M A19 T1
OPH-15
Fig. (63): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolate (A19 T1 10min) using primer OPH-15 M
(DNA marker) = 100 base pairs ladder.
A19 T1 M
OPC-10
Fig. (64): RAPD banding patterns amplified A. niger (A19) and its
obtained isolate (A19 T1 10min) using primer OPC-10 M
(DNA marker) = 1Kb ladder.
- 112 -
T1 A19 M
OPC-20
Fig. (65): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolated (A19 T1 10min) using primer OPC -20 M
(DNA marker) = 1 kb ladder.
A19 T1 M
OPA-20
Fig. (66): RAPD banding patterns amplified for A niger (A19) and its
obtained isolate (A19 T1 10 min) using primer OPA-20 M
(DNA marker) = 1kb
- 113 -
Fig. (67): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolate (A19 T1 10min) using primer OPO-04 M
(DNA marker) = 1Kb.
A19 T1 M
OPB-15
Fig. (68): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolate (A19 T1 10min) using primer OPB-15 M
(DNA marker) = 1kb ladder.
- 114 -
M A19 T1
OPB-05
Fig. (69): RAPA banding patters amplifying for A. niger (A19) and its
isolate (A19 T1 10min) using primer OPB-05M (DNA marker)
= 1kb ladders.
M A19 T1
OPB-07
Fig. (70): RAPD banding patterns amplified for A. niger (A19) &
obtained isolate (A19 T1 10min) using primer OPB-07M (DNA
marker) = 100 base ladder.
- 115 -
M A19 T1
OPB-06
Fig. (71): RAPD banding patterns amplified for A. niger (A19) and its
obtained isolate (A19 T1 10min) using OPB-06 M (DNA
marker) = 1kb ladder.
- 116 -
Table (32), sixteen positive unique marker identified mutant
A19T1 10min amplified by primer OPG-05 at
molecular weight 3458bp, primer OPA-20 at
molecular weight 211, 185bp, primer OPB-07 at
molecular weight 1601, 862, 542, 358, 263 and
193bps and primer OPH-15 at molecular weight
1456, 895, 793, 550, 414, 312 & 217 bp these sixteen
bands found only on A19T1 10min. On the other
hand, twelve unique markers for the wild strain A.
niger A19 amplified by OPB-15 primer at molecular
weight 1211bp, OPB-06 primer at molecular weight
3016 bp, OPC-10 primer at molecular weight 284 bp,
OPB-07 primer at molecular weight 1116, 738, 489,
377 and 214bp and OPH-15 primer at molecular
weight 1011, 731, 487 and 235bps.
From table (33), it can be noticed that the genetic similarity
between two genotypes wild strain A. niger A1 (potent strain) and
its mutant (A1T1 5 min) is 81% and variation is 19%, while the
genetic similarity between wild type strain of A. niger A19 (week
strain) and its mutant A19 T1 10min is 73.6% and variation 26.4%.
- 117 -
Table (32): Positive and negative unique RAPD markers and
their molecular weight for wild A. niger A19 (weak
isolate) and its obtained isolate (A19T1 10 min)
after treatment with uv irradiation.
Primers Approx. band size in bp A19 A19T1 10min
OPG-05 OPB-15 OPB-06 OPA-20
OPC-10 OPB-07
OPH-15
3458 1211 3016 211 185 284 1601 1116 862 738 542 489 377 358 263 214 193 1456 1011 895 793 731 550 487 414 312 235 217
0 1 1 0 0 1 0 1 0 1 0 1 1 0 0 1 0 0 1 0 0 1 0 1 0 0 1 0
1 0 0 1 1 0 1 0 1 0 1 0 0 1 1 0 1 1 0 1 1 0 1 0 1 1 0 1
Presence of a band in a genotype =1 Absence of the shared band in the other genotypes = 0
- 118 -
Table (33): RAPD based genetic similarity (GS) between
genotypes of A. niger: (A1 & A1 T1 5 min) - (A19 &
A19T1 10min).
Genotype A1T1 5 min A19T1 10 min
A1
A19
0.81
0.736
Gs (ij) = 2Nij/ (Ni+ Nj) (Nei and Li 1979).
Gs= genetic similarity index
Ni j = number of bands shared by i and j
Ni = number of bands present in I
Nj = number of bands present in j
Gs (A1 A1T1) = 2 NA1A1T1/ (NA1+ NA1T1)
= 2 x 30/ (39+35)
= 60/74
= 0.81
= 81.1%
Gs (A19 A19 T1) = 2 N A19 A19 T1/ (NA19 + NA19 T1)
= 2 x 39 / (51+55)
= 78/106
= 0.736
= 73.6%
GD = 1-GS
GD = genetic distance
GD A1, A1 T1 = 1-0.18 = 0.19
GDA19, A19 T1 = 1-0.736 = 0.264
- 119 -
Protein profile (SDS-PAGE) was carried out (table 34 &
Figs. 72-76) for assessing any change between A. niger isolates
(wild & mutant strains) i, e A1 & A1,T1 (5 min) - A19 & A19 T1
(10min). Twenty two bands ranged from 243.2 to 30.1kDa were
obtained in wild isolate of A. niger A1 (potent), on the other hand
after uv irradiation (5min), mutant A. niger A1 T1 5min gives
fifteen bands ranged from 235.77 to 31.96KDa.
In A. niger A19 (wild weak strain) showed twelve bands
ranged from 91.12 to 24.78KDa, while after uv irradiation
(10min) in treated isolate A19T1 10 min showed sixteen bands
ranged from 100.74 to 21.15KDa.
- 120 -
Table (34): SDS-PAGE for (A1 &A1 T1 5 min and A19 & A19 T1
10 min.).
Lane Number
Band Number
Relative Front
Mol. Wt. KDa
Peak OD
Average OD
Marker
1 1 0.195 250 99.693 60.645
1 2 0.276 130 39.525 22.645
1 3 0.352 95 42.977 25.01
1 4 0.547 72 19.883 12.309
1 5 0.655 55 14.397 7.915
1 6 0.798 36 74.681 50.151
1 7 0.958 22 72.77 50.4
A1 T1 5 min
2 1 0.202 235.773 26.977 17.367
2 2 0.234 182.171 29.821 15.674
2 3 0.338 97.873 96.187 69.053
2 4 0.361 93.214 67.105 59.868
2 5 0.383 90.436 45.358 33.877
2 6 0.451 82.363 83.451 56.348
2 7 0.497 77.386 19.315 15.613
2 8 0.55 71.736 39.961 28.765
2 9 0.595 63.963 20.646 11.268
2 10 0.637 57.418 35.529 25.226
2 11 0.671 52.263 18.735 12.452
2 12 0.745 42.049 42.864 32.169
2 13 0.769 39.302 25.163 16.795
2 14 0.817 33.973 28.233 19.969
2 15 0.838 31.964 27.821 17.347
- 121 -
Table (34): (Cont.)
Lane Number
Band Number
Relative Front
Mol. Wt. KDa
Peak OD
Average OD
A1
3 1 0.198 243.228 19.121 12.012
3 2 0.237 173.532 15.556 10.148
3 3 0.338 99.110 103.210 65.743
3 4 0.362 93.081 73.502 64.481
3 5 0.380 90.644 47.650 37.891
3 6 0.403 87.707 18.646 12.536
3 7 0.446 82.871 86.763 51.680
3 8 0.495 77.446 18.708 12.519
3 9 0.546 72.244 47.510 39.434
3 10 0.563 69.225 15.724 9.384
3 11 0.588 65.036 12.296 10.051
3 12 0.610 61.410 9.132 5.359
3 13 0.632 58.183 31.296 22.432
3 14 0.644 56.633 24.72 21.014
3 15 0.663 53.569 26.027 23.172
3 16 0.670 52.444 19.673 15.715
3 17 0.734 43.324 39.428 31.337
3 18 0.758 40.573 24.545 13.209
3 19 0.803 35.559 32.658 24.659
3 20 0.820 33.65 38.233 32.818
3 21 0.830 32.635 36.280 29.192
3 22 0.856 30.134 20.572 12.561
- 122 -
Table (34): (Cont.)
Lane Band Relative Mol. Wt. Peak Average
Number Number Front KDa OD OD
A19 T1 10min
4 1 0.334 100.74 44.953 33.827
4 2 0.36 93.352 46.105 38.476
4 3 0.375 91.469 40.654 31.026
4 4 0.439 83.677 33.673 24.112
4 5 0.451 82.311 30.023 23.159
4 6 0.54 72.987 13.304 8.649
4 7 0.593 64.262 14.782 6.193
4 8 0.635 57.587 23.222 15.373
4 9 0.662 53.746 18.875 13.385
4 10 0.72 45.318 18.272 11.959
4 11 0.743 42.854 25.743 18.948
4 12 0.765 40.693 13.331 8.78
4 13 0.808 35.854 21.529 12.773
4 14 0.819 34.505 27.362 21.497
4 15 0.839 32.25 25.599 18.881
4 16 0.973 21.15 109.716 80.623
A19 5 1 0.378 91.126 30.331 19.856
5 2 0.451 82.252 21.304 11.502
5 3 0.528 74.031 6.84 3.453
5 4 0.55 71.425 5.397 2.002
5 5 0.594 62.989 8.63 2.804
5 6 0.633 58.028 12.556 7.514
5 7 0.676 52.183 21.973 16.201
5 8 0.749 43.479 32.506 26.213
5 9 0.779 40.573 42.3 35.956
5 10 0.832 33.92 43.537 37.267
5 11 0.86 30.238 25.529 14.321
5 12 0.924 24.782 39.506 25.148
A1 : A. niger (potent wild isolate) A1 T1 : A. niger A1 after 5min uv irradiation A19 : A. niger (weak wild isolate) A19 T1 : A. niger A19 after 10min uv irradiation
- 123 -
Fig. (72): Marker Lane Profile
- 124 -
Fig. (73): Lane A1 T1 Profile.
- 125 -
Fig. (74): Lane A1 Profile.
- 126 -
Fig. (75): Lane A19 T1 Profile.
- 127 -
Fig. (76): Lane A19 Profile.
- 128 -
- 129 -
Discussion
Citric acid (a tricarboxylic acid) has a broad use in
preparation of numerous industrial products such as food,
pharmaceutical, chemicals and as a cleaning agents (Lotfy et
al., 2007; Kim et al., 2006 and Ghassempour et al., 2004). A.
niger has been the organism of choice for citric acid production
due to its ease of handling, ability to use a variety of cheep raw
material and high yield of citric acid (Torres and Garcia 2009;
Papagianni 2007; Bayraktar and Mehmetaglu, 2000; Mourya
and Jauhri, 2000; El-Holi 1999; Gutierrez-Rojas, 1995 and
Gupta and Sharma 1994).
In recent years a considerable interest has been shown in
using agriculture products and their residues as alternative sources
of carbon such for citric acid production by A. niger (Alagarsamy
and Nallusamy 2010; Rodrigues et al., 2010; Niamul, et al.,
2009; Darani and Zoghi 2008; Ali 2006; Prado et al., 2005;
Kumar et al., 2003 and Soccol 2001). These residues are very
well adapted to solid state cultures due to their cellulosic and
starchy nature, there has been an increasing trend towards
efficient utilization of these residues, besides being a form of
reducing environmental concerns (Soccol and Vandenberghe
2003).
- 130 -
In this study twenty strains of A. niger were isolated from
different sources, screened for their capacity to produce citric
acid, all the strains were able to produce citric acid in different
quantities at different time intervals i.e., 4, 8, & 12 days on
indicator medium. The best incubation period for production for
all isolates was 12 days. Citric acid productivity was obtained by
all strains under the study when using different concentration (5,
10, 15, 20 and 25%) of four carbohydrate by-products (maize
straw, potato solid wastes, sugar beet pulp and molasses) when
each used alone without any additions after 12 days incubation
and the production enhanced when the fermentation medium
amended with same concentrations of the above mentioned
substrates.
Type and concentration of carbohydrate by-product affect
the production of citric acid by A. niger strains under the study.
Sugar beet pulp & molasses giving the highest yield of citric acid,
while maize straw giving the lowest production by A. niger
strains. Production recorded when using potato wastes was more
than maize straw but lower than sugar beet pulp and molasses.
Increasing the substrate concentration under investigation led to
increase the production for all the isolates with all the
carbohydrate by-products used. Isolates (A1, A4 & A5) recorded
highest yield of citric acid production on different media used,
while A8, A16, A18, & A19 recorded weak production.
- 131 -
In view of other workers, Mehyar et al., 2005 indicated
that sugar from date extract or molasses could be used as
carbohydrate source for citric acid production, strain type, nature
of carbohydrate source and total soluble solids (TSS) has a
significant effect on citric acid production by A. niger.
Vandenberghe et al., 2004 compare citric acid production from
cassava bagasse and two other substrates (sugar cane bagasse &
coffee husk) in SSF using a culture of A. niger, cassava bagasse
giving the highest yield of citric among the tested substrates. Lu
et al., 1995 reported that kumara (Ipomoea batatas) and taro
(Colocasia esculenta) were excellent substrates for citric acid
production by solid substrates fermentation using A. niger,
conversely potato (Solanum tuberosum) was a poor substrate
although it supported profuse growth. Zafiris et al., 1994
examined three strains of A. niger for citric acid production on
orange processing wastes and found that strain NRRL 599 gave
the greatest amount of citric acid. About substrate concerning
concentrations, Mourya and Jauhri 2000 claimed that increasing
substrate concentrations (maize starch hydrolysate) gave
corresponding increase of citric acid by A. niger.
A. niger is one of the best known citric acid producers, it is
used for industrial scale production, in spite of the large number
of producer strains available, it is still important to generate
strains with advantages characteristics such as enhanced citric
- 132 -
acid production and increase rate of fermentation (Conte and
Marine 2003; Rohr et al., 1992).
Strains with superior characters such as enhanced citric
acid production are hope for investigators, the most frequently
used method for enhanced citric acid production is induction by
uv irradiation (Rodrigues et al., 2010). Since uv rays are
absorbed by Purine and Pyrimidines, making them reactive,
inducing mutations (Griffiths et al., 2006 and Zaha, 2003),
therefore mutations induced by uv can randomly provide a strain
with a higher capacity of citric acid production when compared to
the parent strain for citric acid production (Rodrigues et al.,
2010).
The most potent strains of A. niger for citric acid
production in this study obtained by isolates A1, A4 and A5 on the
different used media. These selected parental isolates were
exposed to uv light irradiation and γ-rays as a mutagenic agents as
a trail for improving their capacity for citric acid production. uv
treatment affected positively in citric acid production for treated
ones after 12 days incubation, molasses at 25% conc. only was the
best carbohydrate by product when added to the basal medium for
growing the obtained isolates after uv treatment, followed by
sugar beet pulp, while lowest production recorded with maize
straw, treated isolates growing on indicator medium and basal
- 133 -
medium with potato solid wastes almost equal, both better than
with basal medium with maize straw but lowest than basal
medium amended with molasses.
Production of parental isolates A1, A4, A5, on basal medium
amended with 25% molasses were 1.22, 1.04, 1.20 (mg/ml)
respectively after 12 days incubation while the yield ranged from
(5-5.62mg/ml) for seventeen (17) ones obtained (treated) from
parent A1 after uv irradiation (2, 5, 10, 15 & 20min) with
increases reached to 4.5 fold. As for A4 (11) eleven obtained ones
recorded 2 fold increase in production when compared to the
parental isolate (non-treated), while 9 (nine) derivatives produced
2 fold increase from A5.
Asad-ur-Rehman et al., 2003, Ikram-ul-Hag et al., 2001,
Abou-Zeid and Ashy 1984 & Khan et al.,1970 reported that
molasses a by-product of sugar industry has been a substrate of
choice for citric acid production, molasses medium as the best
fermentation medium for enhanced and consistent yield of citric
acid by A. niger strains. Ali et al., 2001; & Mattey and Allan
1990 found that the production of citric acid by A. niger is one of
the most commercially utilized examples of fungal overflow
metabolism.
Several investigators utilized uv irradiation for mutations
induction from A. niger for citric acid production. The mutant
- 134 -
strains might show several fold increase in citric acid production
as compared to wild type cultures (Lotfy et al.,2007; Ikram et
al.,2004; Asad-ur-Rehman et al.,2003; Conte and Marine
2003; Ikram et al.,2001; Rugsaseel et al.,1993 and Hamissa et
al.,1992). Lotfly et al., 2007 obtained uv mutant of A. niger in
citric acid production approximately 2-3 fold increase when
compared the parental wild type strain. UV irradiation,
ethylmethane sulfonate (EMS) and acridinc (Ao) were used to
induce citric acid over production mutants in A. niger UMIP
2564, among 15, eight of the mutant derivatives were improved
with respect to citric acid production from sucrose in batch
cultures, maximum product yield was recorded by W5 a stable uv
mutant with approximately 3.2 fold increase when compared to
wild type strain.
Ikram ul-Hag et al., 2004 subjected A. niger conidial
suspension of A. niger to uv induced mutagenesis and found that
among 3 variants, GCM was found to be a better producer of
citric acid. Mourya and Jauhri 2000 found that uv was effective
in increasing citric acid by A. niger on starch hydrolysate.
Rugsaseel et al., 1993 obtained mutants with enhanced citric acid
production from soluble starch induced from A. niger WU-2223
after uv irradiation. Hamissa et al., 1992 showed that uv
treatment resulted in the development of 31 isolate of A. niger in
citric acid production from beet molasses.
- 135 -
In this study γ-rays was less effective than uv light
irradiation in increasing citric acid production when treated
selected strains A1,A4 and A5. Increase 2 fold on indicator
medium & fermentation medium amended with molasses at 25%
conc. for some derivative ones at does form 50-200Gy as
mentioned in the results, maximum production obtained after γ-
rays ranged from 2-2.4mg/ml. Positive results were obtained with
γ-rays by Alani et al., 2007 who selected mutant G4 from a strain
of A. niger after four rounds of γ-ray irradiation, the parent strain
produced 30g/kg citric acid, while the mutant produced 60g/kg
from carob under solid-state fermentation. Parvez et al., 1998
studied citric acid production from sugar cane molasses by A.
niger NIAB280 in a batch cultivation process, mutant strain
showing resistance to 2-deoxyglucose in vagals medium
containing molasses as a carbon source were induced by γ-
irradiation, among mutant strains, strain RP7 produced 1.5 fold
improvement than parental strain. Begum et al., 1990 induced
mutants of A. niger in different carbohydrate media for citric acid
fermentation by γ-rays. Golubtsova et al., 1978, 1976 & 1972
reported that the most active and resistant mutants for citric acid
production by A. niger were produced at the does of 10-50 krad
(100-500Gy) of γ-rays.
- 136 -
Pronounced increase in citric acid production reached to
8.5 fold recorded in this study for derivatives ones (treated) T1
and T2 (10 min) of parental weak isolate A19 when exposed to uv
treatment as compared on indicator medium. Production of the
parental strain A19 on indicator medium was 0.60 mg/ml, it
reached to 5.35 mg/ml for A19T1 after 10 min of uv irradiation.
Mourya & Jauhri 2000 and Kunar et al., 1989 observed
variation in citric acid production of various mutants of A. niger
with sucrose as substrate, attributed this effect to the higher
glycolytic activites of these strains subsequent finding on
activities of the two glycolytic enzymes (hexokinase &
phosphofructokinase fructokinase) lent further support to this
explanation that a citric acid accumulation requires a higher
glycolotic activity. Since it is unlikely that mutations have
affected the structural genes for both the enzymes, a regulatory
gene may probably be altered, therefore it is quite possible that an
increase in glycolytic capacity of A. niger increase the ability of
these strains to accumulate citric acid.
PCR is one of the most popular tools in molecular
diagnosis. It can amplify DNA samples to a detectable signal
level within a short period of time, in theory a DNA product can
be amplified and doubled in each cycle, however, primer-diamer
and GC-rich regions of the template and the PCR system’s
heating/coaling ratio may interfere with the efficiency of the PCR
- 137 -
(Li et al., 2005 and Brownie et al., 1997). PCR amplification can
be performed directly on various microbial cultures for
filamentous fungi and yeast, prior isolation of DNA is often
preferred (Plaza et al., 2004 and Liu et al., 2000).
Data in the study revealed that RAPD analysis using PCR
successeded to differentiate between wild strain of A. niger
A1(potent isolate) and the mutant one A1 T1 (5min) after uv
irradiation-in addition to the wild strain A19 (weak isolate) and its
mutant A19 T1 (10min) after uv irradiation. Genetic similarities
between these genotypes. i.e., (A1 & A1 T1 5 min) – (A19 & A19 T1
10min) were 81.0 & 73.6% respectively, while the variation
obtained were 19.0& 26.4% respectively. Protein profile for A1
(wild potent isolate) indicated 22 bands from 243.2 to 30.1 KDa,
there were 15 bands for the obtained mutant A1 T1 5min ranged
from 235.27 to 37.96KDa. As for A19 (wild weak isolate) 12
bands were obtained ranged from 91.12 to 24.78 KDa, while 16
bands were obtained for its mutant A19 T1 (10min) ranged from
100.74 to 21.15KDa.
Mutagenic effect of uv irradiation induced these variations.
It can be noticed that variation between A19 & its mutant (26.4%)
higher than between A1 & its mutant (19%), this may explained to
the fact that citric acid production for A19 T1 10min was 9 fold
over the production of the wild isolate A19, while the production
of the mutant A1 T1 (5min) was 4 times higher than its wild A1.
- 138 -
Vossen et al., (1994) pointed to the applicability of RAPD
markers for identification purposes. The technique is based on the
amplification of random DNA sequences by polymerase chain
reaction (PCR) using arbitrary primers, they optimized the
reaction components and evaluated several primers, the RAPD
assay clearly showed comparative banding patterns for strains
within the same species. This assay enabled to discriminate
between Zygo saccharomyces bailii and rauxii. Within
Saccharomyces cerevisiae species two distinct groups of RAPD-
patterns have been found indicating the possibility to distinguish
between varieties.
- 139 -
Summary
Twenty strains of Aspergillus niger were isolated from
different sources, and screened for their capacity to produce citric
acid on indicator medium and different carbohydrate by-products
i.e. maize straw, potato solid wastes, sugar beet pulp and
molasses. The most potent strains for production were found to be
three strains, out of twenty (A1, A4 & A5) while A8, A16, A18 &
A19, were recorded weak production on indicator medium which
contain different four carbohydrate by-products under the study.
The chosen isolated isolates A1, A4 & A5 were exposed to
uv irradiation for different periods i.e., 2, 5, 10, 15, 20, 25 & 30
minutes & to different does of γ-rays (25, 50, 100 & 200)Gy as a
mutagenic agents in a trail for improving their capacity for citric
acid production, the obtained results can be summarized in the
following:-
1. Type and concentration of carbohydrate by-products under
the study affect citric acid yield by Aspergillus niger strains.
2. Increasing carbohydrate by-product concentration led to
increase the production of citric acid, the best concentration
for production was 25% for maize straw, potato solid wastes
(peels), sugar beet pulp and molasses.
- 140 -
3. Citric acid productivity were obtained by all isolated strains
when using different concentration of the four carbohydrate
by-products when each used alone without any additions
after 12 days incubation and the yield enhanced when the
fermentation medium amended with the same concentrations
of the mentioned substrates.
4. Sugar beet pulp and molasses giving the highest yield by
Aspergillus niger strains under the study while maize straw
giving lowest production. The yield on indicator medium and
potato solid wastes almost equal. Both better than maize
straw but lower than sugar beet pulp and molasses.
5. UV treatment affect positively in citric acid production by
selected isolates of A. niger growing on different
carbohydrate by-products. Molasses was the best
carbohydrate by-product for citric acid production by treated
ones when added at 25% to the basal medium after 12 day
incubation followed by sugar beet pulp, while the lowest
yield recorded with maize straw. Production by treated
isolates after uv treatment growing on indicator medium and
basal medium with potato solid wastes almost equal, both
better than with maize straw but lower than molasses and
sugar beet pulp.
- 141 -
6. Production of parental isolates A1, A4 & A5 on basal medium
with 25% maize straw were: 0.73, 0.70 & 0.85(mg/ml)
respectively after 12 days incubation. Maximum yield from
the treated (obtained ones) after u.v. irradiation were: 1.50,
1.45 by A4 T2 (5min) & A4 T1 (10min) respectively with
increase 2 fold.
7. Yield of citric acid by parental isolates A1, A4, & A5, on basal
medium with 25% potato solid wastes were: 0.94, 0.91 &
0.95 (mg/ml) respectively after 12 days incubation, while
maximum yield by the treated ones after uv treatment were
1.85, 1.84 & 1.78 (mg/ml) by A1 T1 (5min), A4 T2 (5min) A1
T3 (5 min) respectively.
8. On basal medium amended with 25% sugar beet pulp, the
production of parental isolates A1, A4, & A5 after 12 day
incubation were: 1.60, 1.50 & 1.51(mg/ml) respectively after
12 days incubation, while maximum yield by the treated ones
after u.v. treatment reached to 3.16 & 2.82 (mg/ml) with
increase 2 fold by A1 T1 (5min) & A1 T5 (5min) respectively.
9. The production of citric acid on basal medium amended with
25% molasses after 12 days for the parental isolates A1, A4 &
A5 were: 1.22, 1.04 & 1.20 (mg/ml) respectively while the
yield ranged from 5-5.62mg/ml for 17 ones obtained (treated
with uv treatment) form the parental A1 after exposure times
- 142 -
2, 5, 10, 15 & 20 minutes with increase reached to 4.5 fold,
maximums yield with A1 T1 (5min) was 5.62mg/ml.
10. UV treatment was more effective than γ-rays in producing
number of obtained isolates of A. niger yielding more
production of citric acid than the parental strains. On basal
medium amended with 25% molasses, increase 2 fold in
production of citric acid for A1 after γ-rays treatment after 12
days incubation by A1T1 (100Gy), the yield reached to 2.40
(mg/ml) while it was 1.20 (mg/ml) for the wild isolate A1.
11. Increase 2 fold in citric acid production on indicator medium
for the treated A. niger after γ-rays treatment after 12 days
incubation in derivatives A1(T1 50 Gy; T1, T2, T3 & T4 100
Gy; T1 & T2 200 Gy), A4(T1, T2 & T3 100Gy), A5(T1 & T2
100 Gy; T1 & T2 200 Gy).
12. Slight increase in citric acid production in few treated ones
on basal medium with 25% maize straw when compared with
the parental isolates (untreated with γ-rays).
13. When the basal medium amended with 25% potato solid
wastes, maximum citric acid production after 12 days
incubation and γ-rays treatment were : 1.80, 1.65, 1.60 &
1.63 (mg/ml) by A1(T1, T2, T3 & T4 after exposed to 100Gy)
with increase 1.5 fold, no increase in production obtained in
- 143 -
the derivatives for A4 & A5 after all doses of γ-rays (25, 50,
100 & 200 Gy).
14. Slight increase in citric acid production for some obtained
ones on basal medium with 25% sugar beat pulp when
compared with the parental isolates (untreated with γ-rays).
15. When the basal medium amended with 25% molasses,
increase in production for ten (10) obtained ones for A1 after
treatment with γ-rays. 2 fold recorded after 12 days
incubation by A1 (T1 100 Gy) & 1.5 fold for T1 25 Gy; (T1,
T2, T3 ) 50 Gy; (T2, T3, T4) 100 Gy & (T1, T2) 200 Gy. Also
increases in production reached to 1.5 folds for some
obtained ones in this carbohydrate by-product by A4 & A5
strains.
16. Production of parental weak isolates A8, A16, A18, & A19 on
indicator medium after 12 days incubation were 0.74, 0.70,
0.69 & 0.60 (mg/ml) respectively. Interestingly, increase of
production fold reached to 5, 6, 7, 8 & 9 folds recorded after
uv irradiation. Maximum production of citric acid were:
3.60, 5.20, 5.20 & 5.35 (mg/ml) by A8T1 (10min), A16T1
(10min), A18T1 (10min) & A19T1 (10min) respectively.
17. RAPD-PCR analysis revealed that five positive unique
markers amplified by OPO-14 primer at molecular weight of
- 144 -
4245bp, OPA-18 primmer at molecular weight of 811bp, and
OPA-04 primer at molecular weight 572, 219 and 135bp
identified the mutant A1T1 5min, these five bands found only
on A1T1 5min, while in the wild isolate A1 there were nine
markers amplified by OPO-02 primer at molecular weight of
2115 and 811bp, OPO-14 primer at molecular weight pf 578
& 325bp, OPC-14 primer at molecular weight of 548bp,
OPA-05 primer at molecular weight 604 & 480bp and OPB-
05 primer at molecular weight 453 & 272bp.
18. Sixteen positive unique markers amplified by primer OPG-
05 at molecular weight 3458bp, primer OPA-20 at molecular
weight 211, 185pb, primer of OPB-07 at molecular weight
1601, 862, 542, 358, 263 and 193 bp and primer OPH-15 at
molecular weight 1456, 895, 793, 550, 414, 312, 217 bp
identified the mutant A19 T1 (10min.) while in the wild
isolate A19 twelve unique markers were found amplified by
OPB-15 primer at molecular weight 1211bp, OPB-06 primer
at molecular weight 3016bp, OPC-10 primmer at molecular
weight 284bp, OPB-07 primer at molecular weight 1116,
738, 489, 377 and 214bp1 and OPH-15 primers at molecular
weight 1011, 731, 487 and 235 bps.
19. The genetic similarity between two genotypes: wild strain A1
and its mutant A1 T1 5min is 81% and variation is 19% while
- 145 -
the genetic similarity between wild type strain of A. niger
A19 and its mutant A19 T110 min is 73.6% and variation
26.4%.
20. In protein profile (SDS-PAGE) twenty two bands ranged
from 243.2 to 30.1 KDa were obtained in A. niger (A1)
(Parental potent isolate) on the other hand after uv irradiation
(5min) mutant A. niger A1T1 (5min) have fifteen bands
ranged from 235.77 to 31.96kDa.
21. A. niger A19 (parental weak isolate) showed twelve bands
raged from 91.12 to 24.78kDa, while after uv irradiation
(10min) mutant obtained isolate A19T1 (10min) showed
sixteen bands ranged from 100.74 to 21.15kDa.
- 146 -
- 147 -
CONCLUSION
• Sugar beet pulp and molasses giving the highest yield of CA by A. niger isolates while maize straw giving the lowest CA production. The yield on synthetic medium and potato solid wastes almost equal. Both better than maize straw but lower than sugar beet pulp and molasses.
• UV treatment after positively in CA production by selected isolates of A. niger growing on different carbohydrate by-products.
- Maximum yield of CA by treated isolates of A. niger recorded increase 2 folds on basal medium amended with maize straw or sugar beet pulp with 25% conc.
- Slight increase in CA production by treated isolates of A. niger growing on basal medium amended with potato solid wastes with 25% conc.
- Maximum production of CA basal medium amended with 25% molasses by treated isolate (A1T1 5min.) reached to 4.5 folds than parental isolate (Al).
- Production of CAby weak isolate (A19T1 10 min.) reached to 8.5 folds than wild isolate (A19).
• Gamma ray was less effective than uv irradiation in increasing CA production by treated isolates. The highest production recorded increase 2 folds by treated isolates on synthetic medium and basal medium amended with molasses with 25% conc. at 100 Gy.
• The genetic similarity between two genotypes: wild strain A1 and its mutant A1T1 (5min) is 81% and variation is 19% while the genetic similarity between wild type strain of A. niger A19 and its mutant A19T1 (10 min.) is 73.6% and variation 26.4%.
- 148 -
Recommendation :
• All the isolates of A. niger have the ability to produce CA in
different quantities at different time intervals.
• The most potent isolates A1, A4 & A5 while the weak
isolates A8, A16, A18 & A19.
• The best incubation periods for CA production by isolates
was 12 days.
• Type and concentration of carbohydrate by-products affect
CA production by isolates of A. niger.
• UV treatment was more effective than γ-rays. It can
randomly provides mutant with hyperproduction for CA
when compared to the wild strain.
• UV irradiation affect positively in CA production by weak
producer of A. niger (A19T1 10 min.) more than active
producer (A1T1 5 min.).
• CA production by A1T1 5 min of A. niger on maize straw,
potato solid wastes, sugar beet pulp and molasses after 12
days incubation 1.30, 1.85, 3.16 and 5.62 mg/ml respectively
by CA production by A19T1 10 min of A. niger on the same
substrates after 12 days incubation 2.05, 2.83, 3.80 & 6.10
mg/ml respectively.
- 149 -
References
Abou-Zeid A.Z. and Ashy M.A. (1984): Production of citric acid:
A review. Agricultural Wastes, 9:51-76.
Adham N.Z. (2002): Attempts at improving citric acid
fermentation by Aspergillus niger in beet molasses
medium. Bioresource Technology, 48:97-100.
Alagarsamy K. and Nallusamy S. (2010): Citric acid production
by koji fermentation using Bio-resource Technology,
101(14): 5552-5556.
Alani F.; Moo-Yong M.; Anderson W. and Bataine Z. (2007):
Optimization of citric acid production from a new strain
and mutant of Aspergillus niger using solid state
fermentation. Food Biotechnology, 21: 169-180.
Ali S., (2006): Application of Kaolin to improve citric acid
production by a thermophilic Aspergillus niger. Applied
Microbiology and Biotechology, 37: 4, 755-762.
Ali S., Ul-Haq I, Qadeer M.A. and Iqbal J. (2001): Biosynthesis
of citric acid by Locally isolated Aspergillus niger using
sucrose salt media. J. Biol. Sci., 1: 178-181.
Arzumanov T.E., Shishkanova N.V. and Finogenova T.V.
(2000): Biosynthesis of citric acid by Yarrowia lipplytica
repeat batch culture on ethanol. Applied Microbiology
Biotechnology 53: 525-529.
- 150 -
Asad-ur- R.; Sikander A. and Ikram-ul. H. (2003): Selection of
fermentation for citric acid. Bioreactor Bio-technology, 2:
178-184.
Asad-ur-R.; Sikander A. and Ikram-ul. H. (2003): Phosphate
limitation for enhanced citric acid fermentation using
Aspergillus niger mutant UV-M9 on semi-pilot- scale
Pakistan J. of Biological sciences, 6 (14) 1247-1249.
Baig M.M.V., Baig M.L.B. and Majeda Y. (2004):
Sccharification of banana agro-waste by cellulolytic
enzymes. African Journal of Biotechnology. 3, 9, 447-
450.
Barrington S. and Woo K. J. (2008): Response surface
optimization of medium components for citric acid
production by Aspergillus niger NRRL 567 grown in peat
moss. J. of Biotechnology, 99: 368-377.
Bayraktar E. and Mehmetaglu U. (2000): Production of citric
acid using immobilized conidia of Aspergillus niger.
Appl. Biochem. Biotechnol., 87: 117-125.
Begum A.; Choudhurg N. and Islam M. (1990): Citric acid
fermentation by gamma ray induced mutants of
Aspergillus niger in different carbohydrate media Journal
of fermentation and bioengineering, 70 (4): 286-288.
Begum A.A.; Choudhury N. And Islam M.S. (1991): Kinetic
studies on citric acid production by gamma ray induced
- 151 -
mutant of Aspergillus niger. Journal of Bangladesh
Academy of science., 15 (1): pp. 1-6.
Beli I. and Odian G. (2000): Organic and Biochemistry
connecting chemistry to your life. W.H. Freeman and
Company New York.
Black W.C.; Du Teau N.M.; Puterka G.J.; Nechols J.R. and
Pettorini J.M. (1992): Use of the random amplified
polymorphic DNA polymerase chain reaction (RAPD-
PCR) to detect DNA polymorphism's in aphids. Bull. Ent.
Res. 82: 151-159.
Bouchard E.F and Merritt E.G. (1979): In Grayson, M. ed.,
Kirk- Othmer, Encyclopedia of chemical technology, Vol.
6, 3rd ed., John Wiley and Sons, Inc., New York, 150-179.
Brownie J.; Shawcross S.; Theakev J.; Whitcombe D.; Ferrie
R.; Newton C. and Little S. (1997): The elimination of
Primer-diamer accumulation in PCR. Nuchic Acids Res.,
25: 3235-3241.
Chopra C.L.; Gaind C.N.; Quazi GN.; Chaturvedi S.K. and
Somal P. (1983): Production of citric acid by submerged
fermentation. I optimization of parameters. Research and
Industry, 28: 107-113.
Conte A.P.A. and Marine J.M. (2003): Selection of 5-
fluorocytosine resistant mutants from an Aspergillus niger
- 152 -
citric acid producing strain. Braz. J. Microbiology, 34: 1-
7.
Crolla A. and Kennedy K.J. (2001): Optimization of citric acid
production from Candida lipolytica Y- 1095 using n-
paraffin. J. of Biotechnology, 89: 27-40.
Darani K. and Zoghi A. (2008): Comparison of pretreatment
strategies of sugarcane bagasse: Experimental design for
citric acid production. Bioresource Technology, 99: 6986-
6993.
Das A. and Nandi P. (1972): Specific effects of mutagens on
Aspergillus niger producing citric acid. Folia
Microbiology, 17: 248-250.
El-Batal A. I.; Khalil A. H. and Mostafa M.M. (1995): Citric
acid production by gamma irradiated A. niger from
treated beet molasses under different fermentation
conditions. Egypt J. Rad. Sci. Appl., 8 (2) 253-266.
El-Batal A.; Khalaf S. and Khalil A. (1995): Upgrading of citric
acid production from cheap carbohydrate sources as
affected by Aspergillus niger and gamma irradiation.
Egpyt. J. Rad. Sci. Applic. 8: 237-252.
El-Holi M. A. (1999): Citric acid production by selected
Aspergillus species using whey as fermentation medium
M. Sc. Thesis, University of Jordan, Amman., Jordan.
- 153 -
El-Sayed S.A. (1986): Production of citric acid by yeast, Ph. D.
Thesis, Faculty of Agriculture Ain Shams University
Egypt.
Fatemi S.S. and Shojaosadati S.A. (1999): Citric acid production
from apple pomace in solid state fermentation. Iran. J.
Chem. Eng., 18: 44-47.
Fiedurk J.B.; Plute J.; Szezodark and Jainroz (1996):
Relationship between citric acid and extra cellular acid
phosphate production by Aspergillus niger. Acta
Biotechnol., 16: 207-213.
Flavera C.P.; Luciana P. and Carlos R.S. (2005): Relation
between citric acid production by solid-state fermentation
from cassava bagasse and respiration of Aspergillus niger
LPB 21 in semi-pilot scale. Brazilian Archives of Biology
and Technology. 48: 1-10.
Francis F.J. (2000): Encyclopedia of Food Science and
Technology. 2nd ed., Vol. 1 John Wiely and Sons, Inc.
New. York.
Ghassempour A.; Nojavan S. and Talebpour Z. (2004):
Monitoring of the fermentation media of citric acid by the
Trimethylsilyle derivatives of the organic acids formed. J.
Agri. Food Chem., 52: 6384-6388.
Golubtsova V.M.; Shcherbakova E. Ya.; Lunkovskoya L. Y. and
Ermakova V.P. (1978): Changes in the ratio between
- 154 -
citric acid and oxalic acid in Aspergillus niger under the
action of mutagenic factors. Mikrobiologiya., 48: 1060-
1065.
Golubtsova, V.M., Shcherbakova E. Ya. and Smirnov, V.A.
(1972): Effect of gamma irradiation on variability of
citric acid producing Aspergillus niger. Mikrobiologiya.,
41: 700-703.
Golubtsova V.M. Shcherbakova E.Ya., Smirnov V.A. and
Runkovskaya L. Ya. (1976): Stimulation of the
germination of the conidia Aspergillus niger a citric acid
producer, under the action of mutagenic factors.
Radiologiya., 16: 292-295.
Good D.W., Droniuk R., Law ford R.G. and Feni J.E.(1985):
Isolation and characterization of a Saccharomycopsis
lipolitica mutant showing increased production of citric
acid from canola oil. Can. J. Microbiol., 31: 436-440.
Goulart R.D. and Marin J.M. (2005): Reversion by calcium of a
yeast-like development to the original filamentous form
of the 10v105-fluorocytosine-sensitive mutant of
Aspergillus niger. Barazilian. J. of Microbiology., 36:
301-306.
Griffiths A.J.F.; Wesller S.R.; Lewontin R.C.; Gelbart W.H.,
Suzuki D.T. and Miller J.H. (2006): Introduçao á
- 155 -
genética (8th ed.) Rio de Janeiro-RJ, Brazil: Guanbara-
Koogan.
Gupta S. and Sharma C.B. (1994): Continuous production of
citric acid from sugarcane molasses using a combination
of submerged immobilized and surface stabilized cultures
of Aspergillus niger, KCU 520. Biotechnol. Lett., 16 (6):
599-604.
Gutierrez-Rojas M. (1995): Citric acid and polyals productions
by Aspergillus niger at high glucose concentration in
solid state fermentation on inert support. Biotechnol,
Lett., 17 (2): 219-224.
Hagedorn S. and Kaphammer K. (1994): Microbial biocatalysis
in the generation of flavor and fragrance chemical. Am.
Rev. Microbiol., 48: 773-800.
Hamissa F.A.; El-Abyad A.M.; Abdu A. and Gad S.A. (1992):
Raising potent UV mutants of Aspergillus niger van
Tieghem for citric acid production from beet molasses.
Bioresource Technology, 39: 209-213.
Hang Y.D. and Woodams E.E. (2001): Enzymatic enhancement
of citric acid production by Aspergillus niger from
corncobs. Lebensm-Wiss U- Technology, 34: 484-486.
Hang Y.D. and Woodams, S.E. (1995): Grape pomacea novel
substrate for microbial production of citric acid
Biotechnol. Lett., 7: 253-254.
- 156 -
Higgins I.J.; Best D.J. and Jones J. (1985): Biotechnology
Principles and Applications. Blackwell scientific
Publication, Oxford London. 331-332.
Ikram-ul-Hag; Samina K; Sikander A.; Hamed A.; Qadeer
M.A. and Ibrahim R.M. (2001): Mutation of Aspergillus
niger for hyperproduction of citric acid from black strap
molasses. World J. of Microbiology and Biotechnology,
17: 35-37.
Ikram-ul-Hag; Ali S. and Iqbal I. (2003): Direct production of
citric acid from raw starch by Aspergillus niger Process
Biochemistry, 38: 921-924.
Ikram-ul-Hag; Sikander A.; Qadeer M.A. and Javed I. (2004):
Citric acid production by selected mutants of Aspergillus
niger from cane molasses. Bioresource Technology, 93:
125-130.
Ishaq A.; Sikander A.; Ikram-ul-Hag and Qadder M.A. (2002):
Time course profile of citric acid fermentation by
Aspergillus niger and its kinetic relations. Online J. of
Biological Science, 2(11): 760-761.
Islam M. (1990): Citric acid fermentation by Aspergillus niger:
Induction of high yielding mutants with the help
radiation, combination treatments protoplast fusion and
genetic engineering. IAEA, Vienna (Austria) Final Report
of a coordinated research programme on nuclear
techniques in the improvement of traditional fermentation
- 157 -
practice in developing countries with particular emphasis
on cassava. 1986-1989. Oct. 1990. 149: pp. 141-144.
Islam M.S.; Begum R. and Choudhury N. (1984): Semipilot
scale studies on citric acid fermentation by a gamma-ray
induced mutant of A. niger, Biotechnology Letters, 6(7):
431-434.
Khan M.A.A; Hussain M.M.; Khalique A. and Rahman M.A.
(1970): Methods of citric fermentation from molasses by
Aspergillus niger. Pak. J. Sci. Ind Res., 13 : 439- 444.
Khare S.K.; Jha K. and Gandhi A.R. (1994): Use of agarose-
entrapped Aspergillus niger cells for the production of
citric acid from soy whey. App. Microbiol. Biotechnol. 41
(5): 571-573.
Kim J.W.; Barrington S.; Sheppared J. and Lee B. (2006):
Nutrient optimization for the production of citric acid by
Aspergillus niger NRRL 567 grown on peat moss
enriched with glucose. Process Biochemistry, 41: 1253-
1260.
Kishore D. and Smis K.S. (1993): Production of citric acid by
candida lipolytic Y1095. Effect of glucose concentration
on yield and productivity. Enzyme Microb. Technol., 15:
646-651.
- 158 -
Klrimura K.; Itookiya.; Matsuo Y.; Zhang M. and Usami S.;
(1990): Productions of cellulase and citric acid by
intergenetic fusants obtained via protoplast fusion
between Aspergillus niger and Trichoderma viride. Agric.
Biol. Chemi., 54: 1281-1283.
Kolicheski M.B. (1995): Produção de Ácido Cirtico por
Fermtação no Estado Sólido urilizando como substrato
Bagaço de Mandioca. M.Sc. Thesis, Unverisidade Federal
de Paraná, Brazil.
Kumar D.; Jain V.K.; Skanker D. and Srivastava A. (2003):
Citric acid production by solid state fermentation using
sugarcane bagasse. Process Biochemistry., 38: 1731-
1738.
Kumar D.; Jain V.K.; Skanker G. and Srivastava A. (2003):
Utilization of fruits waste for citric acid production by
solid state fermentation. Process Biochemistry., 38: 1725-
1729.
Kunar G. S.; Grotz M.; Rohr M. and Kubieck C.P. (1989):
Increased citric acid by mutants of Aspergillus niger with
increased glycotic capacity fams. Microbiol., Letters
59:297-300.
- 159 -
Kurbanoglu E.B. (2004): Enhancement of citric acid production
with ram horn hydrolysate by Aspergillus niger.
Bioresource Technoloy., 92: 97-101.
Laemmli U.K. (1970): Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature,
277:680-685.
Li M.; Lin Y. C.; Eu C.C. and Liu H.S. (2005): Enhancing the
efficiency of a PCR using gold nanoparticles. Nucleic
Acids Research, 33 (21): 34-41.
Liu D.; Coloe S.; Baird R. and Pederson J. (2000): Rapid mini-
preparation of fungal DNA for PCR. J. Clin. Microbial
38: 431-474.
Lotfy W.A.; Khaled M.G. and Ehab R.E. (2007): Citric acid
production by a novel Aspergillus niger isolate: II.
optimization of process parameters through statistical
experimental designs. Bioresource Technology, 98: 3470-
3477.
Lotfy W.A.; Khaled M. G. and Ehab R.E. (2007): Citric acid
production by a novel Aspergillus niger isolate I.
Mutagenesis and cost reduction studies. Bioresource
Technology. 98: 3464-3469.
- 160 -
Lu M. Y.; Maddox I.S. and Brooks J.D. (1995): Citric acid
production by Aspergillus niger in solid-substrate
fermentation. Bioresource technology 54: 235-239.
Luciana P.S. Vandenberghe L.P.S; Carlos R.; Soccol C.; Prodo
F.C. and Pandey A. (2004): Comparison of citric acid
production by solid state fermentation in Flask, column,
tray, and drum bioreactor. Applied biochemistry and
Biotechnology. 118: 293-303.
Maddox I.S and Brooks J.D. (1998): Application of a
multiplayer packed bed reactor to citric acid production in
solid state fermentation using Aspergillus niger. Process
Biochem. 33: 117-123.
Marrier J. R. and Boulet M. (1958): Direct determination of
citric acid in milk with an improved pyridine- acetic
anhydride method. J. Dairy Science. 41: 1683-1692.
Mattey M. and Allan A. (1990): Glycogen accumulation in
Apsergillus niger. Trans. Biochem. Soc., 18: 1020-1022.
Mehyar G.F.; Delaimy K.S. and Ibrahim S.A. (2005): Citric acid
production by Aspergillus niger using Date-based
medium fortified with whey and additives. Food
Biotechnology 19: 173-144.
- 161 -
Mourya S. and Jauhri K. S. (2000): Production of citric acid
from starch-hydroysate by Aspergillus niger.
Microbiology Research 155: 37-44.
Mullis K.; Faloona F., Scharf S.; Saiki R.; Horn G. and Erlich
H. (1986): The polymerase chain reaction cold spring
Harbor. Symp. Quant. Biol., 51: 263-273.
Nei M. and Li W.H. (1979): Proc. Natl. Acad. Sci., USA, 76,
5269.
Niamul B.; Zahangir A.; Suleyman A.; Parveen J. and
Abdullah A. (2009): Improvement of Production of Citric
acid from oil palm empty fruit bunches: Optimization of
media by statistical experimental designs. Bioresource
Technology, 100(12): 3113-3120.
Nill K.R., (2002): Glossary of Biotechnology Terms. 3rd ed.,
Library of Congress Card Number 2002017441. pp 52-53.
Ou C.Y.; Moore J.L. and Schocetman G. (1991): Use of
irradiation to reduce false positivity in polymerase chain
reaction. Biotechniques. 10: 442-445.
Pallares J.; Rodriguez S. and Sanroman A. (1995): Citric acid
production in Submerged and solid state culture of A.
niger. Bioprocess Engineering 15: 31-33.
- 162 -
Pandey A.; Soccol C.R.; Rodriguez. Leon J.A. and Nigam P.
(2001): Production of organic acids by solid- state
fermentation in: Solid-state fermentation in bio-
technology-fundamentals and applications. New Delhi:
Asiatech Publishers, pp. 113-126.
Papagianni M. (2007): Advances in citric acid fermentation by
Aspergillus niger biochemical aspects, membrane
transport and modeling. Biotechnol. Adv., 25:244-263.
Parvez S.; Rajoka M.I.; Ahmed M.N.; Latif F.; Shahid R. and
Malik K.A. (1998): Citric acid production from sugarcane
molasses by 2-Deoxyglucose resistant mutant strain of
A.niger. Folia Microbiology, 43:59-62
Pazauki M.; Felse P.A.; Sinha J. and Panda T. (2000):
Comparative study on citric acid production by
Aspergillus niger and Candida lipolytica using molasses
and glucose. Bioprocess Eng. 22 : 353-361.
Pera L. M. and Callieri D. A. (1999): Influence of calcium on
fungal growth and citric acid production during
fermentation of a sugarcane molasses-based medium by a
strain of Aspergillus niger. World J. of Microbiology and
Biotechnology 15 (5): 647-649.
Pewlong W.; Sujittra S. and Chanin P. (2003): Improvement in
citric acid production of A. niger ATCC 11414 by
- 163 -
radiation. 29, congress on science and Technology of
Thailand Golden Jubilee convention Hall, Khon Keam
University (Thailand) 20-22 Oct. 2003.
Pintado J.; Torrado A., Gonzalez M.P. and Murado M.A.
(1998): Optimization of nutrient concentration for citric
acid production by solid-state culture of Aspergillus niger
on polyurethane foams. Enz. Microb. Technol., 9: 149-
156.
Plaza G.A.; Upchurch R.; Brigmon R.L; Whiltman W.B. and
Ulfig K. (2004): Rapid DNA extraction for screening soil
filamentous fungi using PCR amplification polish. Journal
of Environmental Studies, 13(3): 315-318.
Prado F.C. (2002): Desenvolimento de Bioprocessoem escala
semipilato produção de ácido citrico par fermentaçao no
estado solido a partir do bagaço de mamdioca. M.Sc.
Thesis, Universidade Federal do parana, Brazil.
Prado F.C.; Vandenberghe L.P. and Soccol C.R. (2005):
Relation between citric acid production by solid state
fermentation from cassava bagasse and respiration of
Aspergillus niger LPB 21 in semi-pilot scale. Braz. Arch.
Boil. Technol., 48: -1-10.
Rodrigues C.; Vandenberghe.; L.P.; Teodoro J.; Pandey A. and
Soccol C.R. (2010): Improvement on citric acid
- 164 -
production in solid. State fermentation by Aspergillus
niger LPBBC mutant using citric pulp. Appl. Biochem.
Biotechnol., 8(8): 8370-8375.
Rohr M.; Kubicek C.P. and Kominek J. (1983): Citric acid in:
Biotechnology, Vol.3, Ver lag Chemie, Weinheim, P.
Rehm, H.J.; Reed, G. (eds.), 419-454.
Rohr M.; Kubicek C.P. and Kominek J. (1992): Industrial acids
and other small molecules. In Aspergillus: Biology and
Industrial Application eds. Bennett, J.W. and Klich, M.A.
pp 93-131. Stoneham: Butterworth –Heinemann. ISBNO-
75069124-7.
Rojoka M.I.; Ahmed M.N.; Shahid F.L. and Pervez S. (1998):
Citric acid production from sugarcane molasses by
Aspergillus niger. Biologia., 44: 241-253.
Roukas T. (1991): Production of citric acid from beet molasses
by immobilized cells of Aspergillus niger. J. Food
Science, 56 (3): 878-880.
Roukas T.(1998): Citric acid production from carb pod extract by
cell recycle Aspergillus niger ATCC 9142. Food
Biotecnol., 12(1/2): 91-104.
Rugsaseel S.; Kirimura K. and Usami S. (1993): Selection of
mutants of Aspergillus niger showing enhanced
- 165 -
productivity of citric acid from starch in shaking culture.
J. of Fermentation and Bioengineering, 75: 226-228.
Rugsaseel S.l Kirimura K. and Usami S. (1993): Citric acid
production from soluble starch in shaking culture by the
selected mutant of Aspergillus niger. Bull. Sci. and Eng.
Res. Lab. Waseda Univ., 142: 39-46.
Rychlik W.; Spencer W.J. and Rhaods R.E. (1990):
Optimization of the annealing temperature for DNA
amplification in vitro. Nucleic Acid Research, 18: 6409-
6412.
Sarangbin S. and Watanapokasin Y. (1999): Yam bean starch:
Anoval substrate for citric acid production by the
protease-negative mutant strain of Aspergillus niger.
Carbohydrate Polymers 38 (3): 219-224.
Shojaosadati S.A. and Babaripour V. (2002): Citric acid
production from apple pomace in multiplayer packed bed
solid state bioreactor. Process Biochem., 37: 909-914.
Soccol C.R. (1996): Citric acid production by Aspergillus niger
using media containing low concentrations of glucose.
Journal of Scientific and Industrial Research, 55: 358-
364.
Soccol C.R. (2001): Developpement de bioprocedes pour la
valorization post-recolt de produits et saus produits
- 166 -
agricoles tropicaux. Memoire (présente en vue de
L'Habilitation á Diriger de Rcherches) université de
provence. Marseille.
Soccol C.R. and Vandenberghe L.P.S. (2003): Overview of
solid-state fermentation in Brazil. Biochem. Eng., J. 13:
205-219.
Soccol C.R.; Prado F.C.; Vandenberghe L.P. and Pandey A.
(2003): General Aspects in citric acid production by
submerged and solid state- fermentation. Encyclopedia of
Bioresource Technology, edited by Pandey A., 652-664.
New York.
Soccol C.R.; Vandenberghe L.P.; Rodrigues C. and Pandey A.F.
(2006): New perspective for citric acid production and
application. Food Tech. Biotechno., 44: 141-150.
Stanburry P. and Whitaker A. (1984): Principles of fermentation
technology. Toronto: Pergmon Press.1-10.
Torres D. and Garcia J. (2009): Assessment of the homeostasis
mechanisms in Aspergillus niger while in citric acid
producing conditions. New Biotechnology, 25(4): 5325.
Tran C.T. and Mitchell D.A. (1995): Pineapple waste a novel
substrate for citric acid production by solid state
fermentation. Biotechnol. Lett., 17: 1107-1110.
- 167 -
Tran C.T.; Sly I.I. and Mitchell D.A. (1998): Selection of a strain
of Aspergillus niger for the production of citric acid from
pineapple waste in solid- sate fermentation-World. J.
Microbiol. Biotechnol., 14: 399-404.
Tran C.T.l Sly I.I. and Mitchell D.A. (1998): Selection of a strain
of Aspergillus niger. Bio process Engineering, 15: 31-33.
Ul-Hag I.; Ali S.; Qadeer M.A and Iqbal J. (2003): Stimulatory
effect of alcohols (methamol and ethanol) on citric acid
productivity by 2-deoxy D-glucose resistant culture of
Aspergillus niger GCB-47 Bioresearch technology, 86:
227-233.
Vandenberghe L.P.; Soccol C.R; Prado F.C. and Pandey A.
(2004): Comparison of citric acid production by solid
state fermentation in flask, column, Tray, and drum
bioreactors. App. Biochem. Biotechol., 118: 293-303.
Vossen J.M.B.M.; Couto M.B.; Eijsma B.; Jos H.J.; Veld B.J.H.
and Hofstra H. (1994): The potential of molecular
methods for typing and identification of spoilage yeast. 7th
International Congress of Bacteriology and Applied
Microbiology Division. International Union of
Microbiology Societies (IU Ms Congresses 94), pp.460 –
MC-8/20.
- 168 -
Welsh J. and Neclellend M. (1990): Fingerprinting genomes
using PCR with arbitrary primers. Nucleic Acid
Research., 18: 7213-7218.
Williams J.G.K.; Kubelik A.R.; Livak K.J.; Rafalski J. A. and
Tingey S.V. (1991): DNA polymorphisms amplified by
arbitrary primers are useful as genetic markers. Nucleic
Acids Res. 18: 6531-6535.
Xie G. and West T.P. (2006): Citric acid production by
Aspergillus niger on wet corn distillers grains. Letters in
Applied Microbiology, 43:269-273.
Yokoya, F. (1992): Fermentaças citrica, Fundaces Tropical de
Pesquisase Tecnologia "André Tosello" Campinas, São
Paulo, Brozil.
Zafiris G.A.; Tzia C.; Oreopoulou V. and Thomopoulos C.D.
(1994): Fermentation of orange processing wastes for
citric acid production. J. Science of Food and Agriculture,
65: 117-120.
Zaha, A. (2003): Biologia molecular basica (3rd ed.) Porto
Alegre-RS, Barazil: Mercado Aberto.
- 1 -
ا��� ا�����
ـ لس نيجر رجليسبة أ عزله من فطر ) ٢٠(تم عزل ادر مختلفـة ـ مـن مص ـ مقدرتهالقياس indicator)ريك علـي البيئـة الدالـة يت علي إنتاج حـامض الس
medium)قش الذرة ـ قشرة البطـاطس ـ (راتية المختلفة د والمخلفات الكربوهي ـوقد) نجر السكر ومولاس قصب السكرمخلف ب ـتزرـ أح A1, A4, A5زلات الع
هـى ,A8, A16, A18, A19هذه البيئات، بينما كانت العزلات ة علي ـأعلى إنتاجي .الأضعف إنتاجاً لذلك الحامض
للأشعة فـوق البنفسـجية A5, A4, A1عرضت العزلات الفطرية المختارة ولجرعات مختلفـة ) دقيقة ٣٠، ٢٥، ٢٠، ١٥، ١٠، ٥، ٢( لفترات زمنية مختلفة
وذلك بغرض تحسين قـدرتها علـي ) جراى٢٠٠، ١٠٠، ٥٠، ٢٥(اما من أشعة ج . إنتاج حامض السيتريك
: ملخص النتائج المتحصل عليها
، ١٥، ١٠، ٥(أثرت نوعية المخلفات الكربوهيدارتية المستخدمة وتركيزها -١علي إنتاج حامض السيتريك بواسطة عـزلات الأسـبرجلس %) ٢٥، ٢٠
.نيجر
بوهيدراتى يؤدى إلـي زيـادة إنتـاج حـامض زيادة تركيز المخلف الكر -٢هو الأمثل للأربع مخلفات المسـتخدمة % ٢٥الستيريك، حيث كان التركيز
وقد زادت إنتاجية حامض السيتريك عند إضافة نفـس التركيـزات مـن . المخلفات الكربوهيدراتية إلي البيئة الاساسية
ـ -٣ ى انتـاج جميع عزلات الاسبرجلس نيجر المختبرة كانت ذات مقدرة علحمض الستريك عند استخدام المخلفات الكربوهيدارتية الأربعة المـذكورة
- 2 -
عند استخدامها بمفردها بدون أي %) ٢٥ ، ٢٠، ١٥، ١٠، ٥(وبتركيزات . يوم تحضين١٢إضافات بعد
تم الحصول علي أعلى إنتاجية لحامض السيتريك باستخدام بنجـر السـكر -٤ بينما كان A. niger ختبرة لفطرومولاس قصب السكر لجميع العزلات الم
مخلف قش الذرة هو أقـل المصـادر الكربوهيدراتيـة المختبـرة إنتاجـاً للحامض، وكان إنتاج حامض السيتريك بواسطة العزلات المختبرة علـي بيئة البطاطس والبيئة الدالة متساوياً فكلاهما أعلى إنتاجاً من استخدام قـش
.مولاسالذرة ولكن أقل من بنجر السكر وال
أثرت المعاملة بالأشعة فوق البنفسجية إيجابياً في إنتاج حامض السـيتريك -٥بواسطة العزلات المختارة من الأسبرجلس نيجر النامية علـي المخلفـات الكربوهيدراتية الأربع حيث كان مخلف مولاس قصب السكر هو الأفضـل
ـ ة لإنتاج حامض السيتريك بواسطة عزلات الأسـبرجلس نيجـر المعاملعلي البيئة الاساسية وبعد % ٢٥بالأشعة فوق البنفسجية عند إضافته بنسبة
يوم من التحضين يليه مخلف بنجر السكر بينما كان مخلف قش الـذرة ١٢هو الأقل إنتاجية لحامض السيتريك ـ أيضاً بعد المعاملة بالأشـعة فـوق
البطـاطس كانت إنتاجية العزلات المتحصل عليها والنامية علي ةالبنفسجيفكلاهما أقل انتاجاً عـن بنجـر السـكر . والبيئة الاساسية متساوية تقريباً . والمولاس وأعلى من قش الذرة
A1, A4, A5:كانت إنتاجية حامض السيتريك للعزلات الأبوية المختـارة -٦ يـوم مـن ١٢علي التـوالي بعـد ) مل/مليجرام (٠,٨٥و٠,٧٠، ٠,٧٣
من مخلف الذرة بينما % ٢٥مضاف إليها التحضين علي البيئة الاساسية وال علـى التـوالى للعـزلات ) مـل /ملجم (١,٤٥، ١,٥٠كانت أعلى إنتاجية
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A4T1,A4T2 دقائق من الأشعة فوق البنفسجية ١٠، ٥ عند التعرض لفترة .بزيادة قدرها ضعفين
، ٠,٩٤: A1, A4 & A5كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٧يوم من التحضين علي البيئة ١٢لي التوالي بعد ع) مل/ملجم (٠,٩٥و٠,٩١
من مخلف البطاطس بينما كانـت أعلـى % ٢٥الاساسية والمضاف إليها عنـد التعـرض A1T1للعزلات ) مل/ملجم (١,٧٨، ١,٨٥، ١,٨٥إنتاجية دقائق من الأشعة فـوق ) ٥( عند التعرض لفترة A4T2دقائق ، ) ٥(لفترة
. رها مرتينالبنفسجية علي التوالي بزيادة قد
، ١,٦٠ A1, A4 & A5:كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٨ يوم من التحضين علي البيئة ١٢علي التوالي بعد ) مل/ملجم(١,٥١و١,٥٠
من مخلف بنجر السكر بينما كانـت أعلـى % ٢٥الاساسية المضافة إليها عنـد A1T1بزيادة ضـعفين للعـزلات ) مل/ملجم (٢,٨٢، ٣,١٦إنتاجية
دقائق للأشـعة ) ٥(عند التعرض لفترة A1T5دقائق ، ) ٥(تعرض لفترة ال .ةفوق البنفسجي
، ١,٢٢ A1, A4 & A5:كانـت إنتاجيـة العـزلات الأبويـة المختـارة -٩يوم من التحضين علي البيئـة الاساسـية ١٢بعد ) مل/ملجم(١,٢٠و١,٠٤
من مولاس قصب السكر بينمـا كانـت الإنتاجيـة % ٢٥والمضافة إليها عزلة ١٧مل بواسطة /ملجم) ٥,٦٢ - ٥(امض السيتريك تتراوح ما بين لح
، ١٠، ٥، ٢ عند التعرض للأشعة البنفسجية لفتـرة A1متحصل عليها من مـل / ملجـم ٥,٦٢ كان أعلى إنتـاج - ضعف ٤,٥ دقيقة بزيادة ٢٠، ١٥
.دقائق) ٥( عند التعرض لفترة A1 T1بواسطة
أفضل عن المعاملـة بأشـعة جامـا المعاملة بالأشعة فوق البنفسجية كانت -١٠مل / ملجم ٢,٤٠وكانت أفضل النتائج المتحصل عليها من هذه المعاملة هي
- 4 -
يوم من التحضين علي البيئة الدالة المضـاف إليهـا ١٢بزيادة مرتين بعد عند تعرضـها لأشـعة جامـا A1من مولاس قصب السكر للعزلة % ٢٥
مل /ملجم١,٢٠ريك حوالي جراي بينما كان إنتاج حامض السيت ١٠٠بمقدار . الأبويةA1للعزلة
– A1بزيادة انتاجية حامض السيتريك مرتين للعزلات المتحصل عليها من -١١ يوم من التحضين على البيئة الدالة عنـد تعرضـها ١٢العزلة الأبوية بعد عنـد A4 جراى ومن العزلة الابوية ٢٠٠، ١٠٠، ٥٠لأشعة جاما بمقدار
عنـد A5 جراى وعند العزلة الابويـة ١٠٠تعرضها لاشعة جاما بمقدار . جراى٢٠٠ و ١٠٠تعرضها لاشعة جاما بمقدار
أدت المعاملة بأشعة جاما إلى زيادة إنتاجية حامض السـيتريك للعـزلات -١٢ يـوم مـن ١٢ زيادة ضـعيفة بعـد A1, A4 & A5المتحصل عليها من
من مخلف قش الـذرة % ٢٥ضاف إليها التحضين على البيئة الأساسية الم . بالمقارنة بالعزلات الأبوية
زيادة إنتاجية حامض السيتريك مرة ونصف للعزلات المتحصل عليها مـن -١٣A1 يوم من التحضين على البيئة الأساسية المضاف ١٢ العزلة الأبوية بعد
و ١,٦٠، ١,٦٥، ١,٨٠من مخلف البطاطس بزيـاده قـدرها % ٢٥إليها عند تعرضها لأشعة جاما A1(T1, T2, T3 & T4)مل بواسطة / ملجم١,٦٣
جراى بينما لا توجد زيادة لأنتاجية حامض السيتريك للعزلات ١٠٠يمقدار ، ٢٥ عند تعرضها لأشعة جاما بالجرعات A5 و A4المتحصل عليها من
. جراى٢٠٠ و ١٠٠، ٥٠
ك للعـزلات أدت المعاملة بأشعة جاما إلى زيادة إنتاجية حامض السـيتري -١٤ يـوم مـن ١٢ زياده ضـعيفه بعـد A1, A4 & A5المتحصل عليها من
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من مخلف بنجر السكر % ٢٥التحضين على البيئة الأساسية المضاف إليها . بالمقارنة بالعزلات الأبوية
زيادة إنتاجية حامض السيتريك لعشر عزلات متحصل عليها مـن العزلـة -١٥البيئة الأساسية المضاف إليهـا يوم من التحضين على ١٢ بعد A1الأبوية
من المولاس زياده قدرها مرتين عند تعرضها لأشعة جاما بمقـدار % ٢٥ جراى وزيادة قدرها مره ونصف عند تعرضها لأشعة جاما بمقـدار ١٠٠أيضاً زيادة قدرها مره ونصف بواسـطة . جراى ٢٠٠ و ١٠٠، ٥٠، ٢٥
. على نفس البيئةA5 & A4العزلات المتحصل عليها من
لحمـض A8, A16 , A18 & A19كانت إنتاجية العزلات الأبوية الضعيفة -١٦ ١٢علي الترتيب بعد ) مل/ملجم (٠,٦٠ و ٠,٦٩، ٠,٧٠، ٠,٧٤ :السيتريك
يوم تحضين ، بعد المعاملة بالأشعة فوق البنفسجية كانت أعلـى إنتاجيـة ) مـل /ملجـم (٥,٣٥ و ٥,٢، ٥,٢، ٣,٦للعزلات المتحصل عليهـا هـى
-A دقائق ،١٠ عند A16 T1 دقائق، ١٠ عند A8 T1 على التوالى للعزلات
18 T1 دقائق و١٠ عند A19T1 دقائق علي التوالي وبزيادة قـدرها ١٠ عند . مرات٨، ٧، ٦، ٥
استخدمت طريقة من طرق البصمة الوراثية وهي طريقة التكبير العشوائي -١٧يـب لتوصيف التباين بـين التراك ) RAPDs(لقطع من الحامض النووي
) uvللسلالة الأم والسلالة المتحصل عليها بعد المعاملة بأشعة الـ (الوراثية بادئ عشوائي للتقييم الـوراثي ) ١٠(وقد استخدم . على المستوى الجزيئي
DNAعلى المستوى الجزيئ للتكبير العشوائي لقطع من الحامض النووي A1T1 (5 min (وتشير النتائج إلى وجود خمس دلائل إيجابية فريدة للطفرة
OPA-18 ، البادئ ٥٤٢٤عند الوزن الجزئي OPO-14بواسطة البادئ
، ٥٧٢ عند الـوزن الجزئـي OPA-04، البادئ ٨١١عند الوزن الجزئي
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وهذه الخمس حلقات موجودة بهذه السلالة فقط بينمـا العزلـة ١٣٥، ٢١٩عنـد OPO-02فهناك تسعة دلائل فريدة بواسطة البـادئ ) الأصلية(الأم
، ٥٧٣ عند الوزن الجزيئ OPO-14، البادئ ٨١١، ٢١١٥لوزن الجزئي ا عند OPA-05، البادئ ٥٤٨عند الوزن الجزيء OPC-14، البادئ ٣٢٥
، ٤٥٣عند الوزن الجزئـي OPB-05 والبادئ ٤٨٠، ٦٠٤الوزن الجزئ ٢٧٢.
بواسـطة A19 T1 (10min) هناك ستة عشر دلائل إيجابية فريدة للطفرة -١٨عنـد OPA-20، البـادئ ٣٤٥٨عند الوزن الجزئي OPG-05البــادئ
، ٨٦٢، ١٦٠١ عند الوزن الجزئي OPB-07، ١٨٥، ٢١١الوزن الجزئي ، ١٤٥٦عند الوزن الجزئي OPH-15 والبادئ ١٩٣، ٢٦٣، ٣٥٨، ٥٤٢ بينما هناك أثني عشر دلائل فريدة ٢١٧، ٣١٢، ٤١٤، ٥٥٠، ٧٩٣، ٨٩٥
عنـد الـوزن OPB-15دئ بواسطـــة البـا A19للعزلة الفطرية الأم والبـادئ ٣٠١٦ عند الـوزن الجزئـي OPB-06 البادئ ١٢١١الجزئي
OPC-10 البادئ ٢٨٤عند الوزن الجزئي ،OPB-07 عند الوزن الجزئي عند الوزن الجزئي OPA-15، والبادئ ٢١٤، ٣٧٧، ٤٨٩، ٧٣٨، ١١١٦٢٣٥، ٤٨٧، ٧٣١، ١٠١١bp.
الأكثر إنتاجـاً لحـامض ( A1م كانت نسبة التشابه الوراثي بين العزلة الأ -١٩الستريك لفطر الأسبرجلس نيجر والطفرة المتحصل عليها بعـد التعـرض
بينما نسبة % ١٩ونسبة الاختلاف % ٨١ A1 T1 دقائق ٥ لمدة uvلأشعة والطفـرة ) الأقل إنتاجـاً ( لفطر الأسبرجلس نيجر A19التشابه للعزلة الام
A19 مدة عشـرة دقائــق بuvالمتحصل عليها بعد التعرض لأشعة
T1٢٦,٤ونسبة الاختلاف % ٧٣,٦.%
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أثنين وعشرين حزمـة (SDS-PAGE)اظهر الفصل الكهربي للبروتين -٢٠بينما هناك خمسـة ) ٣٠,١KDa – ٢٤٣,٢( بين A1للعزلة الأم ) باندات(
– ٢٣٥,٧٧ بــين A1 T1 (5min)للطفــرة ) بانــدات(عشــر حزمــة ٣١,٩٦KDa .
-٩١,١٢بـين ) بانـدات (أثني عشـر حزمـة A19أظهرت العزلة الأم -٢١٢٤,٧٨KDa للطفـرة ) بانـدات ( بينما هناك ستة عشر حزمـةA19 T1
(10min) ٢١,١٥ -١٠٠,٧٤ بينkDa .