Table (1): Optimization of the fungal biomass and MnO NPs production by studying different aeration/agitation rates and its timing, all the way via controlled batch fermentation modeusing7L bioreactor.
|
Batch runs
|
Airflow (vvm)
|
Agitation speed (RPM)
|
|
B1
|
1- 2.5 vvm/12 h
|
100-800 rpm/6 h
|
|
B2
|
0.1- 0.5vvm/12h
|
100-800 rpm/3h
|
|
B3
|
0.5-3.5vvm/12 h
|
50-250 rpm/ 6 h
|
|
B4
|
0.1-4.5vvm/6 h
|
400 rpm
|
|
B5
|
2vvm
|
50-300 rpm/6 h
|
|
B6
|
Airflow and agitation speeds were adjusted to let the dissolved O2 level did not below 10%
|
|
B7
|
Airflow and agitation speeds were adjusted to let the dissolved O2 level did not below 40%
|
Table (2): Optimization of the fungal biomass and MnO NPs production by studying different feeding strategies, feeding regimesandfeeding volumes through controlled fed-batch fermentation modeusing7L bioreactor.
|
Fed-batch runs
|
Feeding strategies
|
Feeding regimes
|
Feeding volumes
|
|
FB1
|
Whole medium ingredients
|
Constantly
|
4ml/6h
|
|
FB2
|
Exponentially via pulsed feeding
|
1.0 - 16.0 ml/6h
|
|
FB3
|
Wheat bran extract only
|
Constantly
|
10ml/6h
|
|
FB4
|
Exponentially via pulsed feeding
|
2.0 - 40.0 ml/6h
|
Table (3): Antimicrobial efficacy of the biogenic synthesized MnO NPs using some phytopathogenic fungi and bacteria.
|
Phytopathogenic fungi
|
|
Zone of inhibition (mm)
|
Phytopathogenic bacteria
|
|
Zone of inhibition (mm)
|
|
Fusarium solani
|
19.25±3.26
|
Clavibacter michiganensis
|
22.5±1.8
|
|
Fusarium moniliforme
|
17.0±3.5
|
Erwinia carotovora
|
24.75±2.68
|
|
Helminthosporium sp.
|
13.0±2.54
|
Acetobacter pasteurianus
|
30.0±2.45
|
|
Alternaria alternate
|
10.25±3.03
|
Erwinia amylovora
|
34.0±2.69
|
|
Aspergillus flavus
|
24.5±3.20
|
Erwinia persicina
|
28.5±5.4
|
|
Aspergillus niger
|
20.5±2.06
|
|
|
Phytophthora arenaria
|
16.0±2.92
|
Table (4): Screening study to detect the produced phytochemical metabolites into endophytic Trichoderma virens strain EG92 fractions which cultivated using Wheat bran medium.
|
Bioactive metabolites
|
Fungal fractions
|
|
* Extracellular
|
Cytoplasmic
|
|
Alkaloids
|
+ve
|
+ve
|
|
Flavonoids
|
-ve
|
-ve
|
|
Tannins
|
+ve
|
-ve
|
|
Phenols
|
+ve
|
+ve
|
|
Steroids
|
+ve
|
-ve
|
|
Saponins
|
-ve
|
-ve
|
|
Terpenoids
|
+ve
|
-ve
|
|
Total Carbohydrate (μg/ml)
|
21.65
|
14.69
|
|
Total protein (mg/µl)
|
16.3
|
12.65
|
Table (5): Matrix of Plackett-Burman experimental design for optimizing the biomass production of endophytic Trichoderma virens strain EG92, showed the coded and actual values for tested variables besides the experimental and predicted results for dry cell mass weight.
|
Exp.#
|
F1
|
F2
|
F3
|
F4
|
F5
|
F6
|
F7
|
F8
|
F9
|
Dry cell mass weight (g/l)
|
Predicted dry cell mass weight (g/l)
|
|
1
|
+
|
+
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
31.50
|
32.13
|
|
2
|
+
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
+
|
24.60
|
23.53
|
|
3
|
-
|
+
|
+
|
+
|
-
|
-
|
-
|
+
|
-
|
20.30
|
21.11
|
|
4
|
+
|
+
|
+
|
-
|
-
|
-
|
+
|
-
|
+
|
21.50
|
22.13
|
|
5
|
+
|
+
|
-
|
-
|
-
|
+
|
-
|
+
|
+
|
41.30
|
40.23
|
|
6
|
+
|
-
|
-
|
-
|
+
|
-
|
+
|
+
|
-
|
33.32
|
34.13
|
|
7
|
-
|
-
|
-
|
+
|
-
|
+
|
+
|
-
|
+
|
31.25
|
32.06
|
|
8
|
-
|
-
|
+
|
-
|
+
|
+
|
-
|
+
|
+
|
35.09
|
35.72
|
|
9
|
-
|
+
|
-
|
+
|
+
|
-
|
+
|
+
|
+
|
29.36
|
28.11
|
|
10
|
+
|
-
|
+
|
+
|
-
|
+
|
+
|
+
|
-
|
36.40
|
35.15
|
|
11
|
-
|
+
|
+
|
-
|
+
|
+
|
+
|
-
|
-
|
27.30
|
26.23
|
|
12
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
25.30
|
24.05
|
|
Inoculums Age (hr)
|
Agitation (RPM)
|
pH
|
CuSO4 (g/l)
|
Incubation temperature (0C)
|
Wheat bran extract (%)
|
Glucose (g/l)
|
Inoculum’s size (%)
|
MgSO4 (g/l)
|
|
|
(-)
|
24
|
150
|
6
|
0.01
|
25
|
20
|
5
|
1
|
0.05
|
|
(+)
|
48
|
200
|
7
|
0.1
|
30
|
30
|
10
|
4
|
0.1
|
Table (6): Regression statistics and analysis of variance (ANOVA) for Plackett-Burman design.
|
Intercept
|
Coefficients
|
Standard Error
|
t Stat
|
P-value
|
Confidence level
(%)
|
|
29.55
|
0.44
|
67.63
|
1.33863E-08
|
|
|
F1
|
1.67
|
0.49
|
3.42
|
0.018934967
|
98.11
|
|
F2
|
-1.23
|
0.49
|
-2.51
|
0.053978572
|
94.60
|
|
*F3
|
-2.24
|
0.49
|
-4.58
|
0.005954457
|
99.40
|
|
F4
|
-0.87
|
0.49
|
-1.77
|
0.136214818
|
86.38
|
|
F5
|
0.43
|
0.49
|
0.87
|
0.422384502
|
57.76
|
|
*F6
|
4.04
|
0.49
|
8.27
|
0.000422473
|
99.96
|
|
F7
|
0.09
|
0.49
|
0.18
|
0.866144618
|
13.39
|
|
*F8
|
2.86
|
0.49
|
5.85
|
0.002059551
|
99.79
|
|
F9
|
0.75
|
0.49
|
1.53
|
0.186109542
|
81.39
|
|
ANOVA
|
|
df
|
SS
|
MS
|
F
|
Significance F
|
|
Regression
|
9
|
423.30
|
47.03
|
16.43
|
0.00332
|
|
Residual
|
5
|
14.32
|
2.86
|
|
|
|
Regression Statistics
|
|
Multiple R
|
R Square
|
Adjusted R Square
|
Standard Error
|
Observations
|
|
0.98
|
0.97
|
0.91
|
1.69
|
15
|
Table (7): Matrix of Box-Behnken experimental design for optimizing the biomass production of entophytic Trichoderma virens strain EG92, showing the experimental and theoretical values of dry cell mass weight under tested variables.
|
# Trial
|
X1
|
X2
|
X3
|
Dry cell mass weight (g/l)
|
Predicted dry cell mass weight
(g/l)
|
|
1
|
0
|
0
|
0
|
71.20
|
70.77
|
|
2
|
-
|
-
|
0
|
58.18
|
61.27
|
|
3
|
0
|
+
|
+
|
70.27
|
71.84
|
|
4
|
0
|
-
|
+
|
61.74
|
60.85
|
|
5
|
0
|
0
|
0
|
70.05
|
70.77
|
|
6
|
+
|
+
|
0
|
62.68
|
59.58
|
|
7
|
+
|
0
|
+
|
81.19
|
82.71
|
|
8
|
+
|
0
|
-
|
83.63
|
85.85
|
|
9
|
-
|
+
|
0
|
66.56
|
67.21
|
|
10
|
-
|
0
|
+
|
83.97
|
81.76
|
|
11
|
0
|
+
|
-
|
60.93
|
61.81
|
|
12
|
0
|
-
|
-
|
79.18
|
77.61
|
|
13
|
-
|
0
|
-
|
86.89
|
85.36
|
|
14
|
0
|
0
|
0
|
71.05
|
70.77
|
|
15
|
+
|
-
|
0
|
70.99
|
70.34
|
|
pH
|
WBE (%)
|
Inoculum’s size (%)
|
|
|
-
|
4
|
30
|
4
|
|
0
|
5
|
40
|
8
|
|
+
|
6
|
50
|
12
|
Table (8): Regression statistics and analysis of variance (ANOVA) for Box-Behnken experimental design.
|
Intercept
|
Coefficients
|
Standard Error
|
t Stat
|
P-value
|
Confidence level (%)
|
|
70.77
|
1.67
|
42.46
|
1.36792E-07
|
|
|
X1
|
0.36
|
1.02
|
0.35
|
0.738184524
|
26.18
|
|
X2
|
-1.20
|
1.02
|
-1.18
|
0.291086336
|
70.89
|
|
X3
|
-1.68
|
1.02
|
-1.65
|
0.15993542
|
84.01
|
|
*X1X2
|
-4.17
|
1.44
|
-2.89
|
0.034116006
|
96.59
|
|
X1X3
|
0.12
|
1.44
|
0.08
|
0.938873008
|
6.11
|
|
*X2X3
|
6.70
|
1.44
|
4.64
|
0.005640579
|
99.44
|
|
*(X1)2
|
4.86
|
1.50
|
3.24
|
0.023068068
|
97.69
|
|
*(X2)2
|
-11.03
|
1.50
|
-7.34
|
0.000736351
|
99.93
|
|
*(X3)2
|
8.29
|
1.50
|
5.52
|
0.002676051
|
99.73
|
|
ANOVA
|
|
df
|
SS
|
MS
|
F
|
Significance F
|
|
Regression
|
9
|
1139.186386
|
126.5762651
|
15.18585511
|
0.003986807
|
|
Residual
|
5
|
41.6757121
|
8.335142421
|
|
|
|
Total
|
14
|
1180.862098
|
|
|
|
|
Regression Statistics
|
|
Multiple R
|
R Square
|
Adjusted R Square
|
Standard Error
|
Observations
|
|
0.98
|
0.96
|
0.90
|
2.89
|
15
|
Table (9): Matrix of Taguchi experimental design for optimizing the MnO NPs biogenic production using endophytic Trichoderma virens strain EG92 extract.
|
Trail
|
A
|
B
|
C
|
D
|
E
|
F
|
MnONPs (g/l)
|
Predicted MnONPs (g/l)
|
S/N Ratio [dB]
|
|
|
T1
|
1
|
1
|
1
|
1
|
1
|
1
|
289.36±4.21
|
293.58
|
49.23
|
|
|
T2
|
1
|
2
|
2
|
2
|
2
|
2
|
265.36±4.7
|
260.79
|
48.48
|
|
|
T3
|
1
|
3
|
3
|
3
|
3
|
3
|
225.36±2.7
|
228.01
|
47.06
|
|
|
T4
|
1
|
4
|
4
|
4
|
4
|
4
|
195.36±0.13
|
195.23
|
45.82
|
|
|
T5
|
1
|
5
|
5
|
5
|
5
|
5
|
160.45±1.99
|
162.45
|
44.11
|
|
|
T6
|
2
|
1
|
2
|
3
|
4
|
5
|
200.09±0.04
|
200.05
|
46.02
|
|
|
T7
|
2
|
2
|
3
|
4
|
5
|
1
|
280.69±0.18
|
280.87
|
48.96
|
|
|
T8
|
2
|
3
|
4
|
5
|
1
|
2
|
262.36±7.1
|
269.46
|
48.38
|
|
|
T9
|
2
|
4
|
5
|
1
|
2
|
3
|
193.89±1.41
|
195.29
|
45.75
|
|
|
T10
|
2
|
5
|
1
|
2
|
3
|
4
|
226.36±5.55
|
220.81
|
47.10
|
|
|
T11
|
3
|
1
|
3
|
5
|
2
|
4
|
240.24±1.3
|
241.50
|
47.61
|
|
|
T12
|
3
|
2
|
4
|
1
|
3
|
5
|
170.28±2.94
|
167.34
|
44.62
|
|
|
T13
|
3
|
3
|
5
|
2
|
4
|
1
|
247.34±0.89
|
248.15
|
47.87
|
|
|
T14
|
3
|
4
|
1
|
3
|
5
|
2
|
280.25±6.58
|
273.66
|
48.95
|
|
|
T15
|
3
|
5
|
2
|
4
|
1
|
3
|
261.36±0.89
|
262.25
|
48.34
|
|
|
T16
|
4
|
1
|
4
|
2
|
5
|
3
|
225.9±5.7
|
220.19
|
47.08
|
|
T17
|
4
|
2
|
5
|
3
|
1
|
4
|
216.36±7.6
|
208.78
|
46.70
|
|
T18
|
4
|
3
|
1
|
4
|
2
|
5
|
229.23±5.06
|
234.29
|
47.21
|
|
T19
|
4
|
4
|
2
|
5
|
3
|
1
|
315.3±0.19
|
315.11
|
49.97
|
|
T20
|
4
|
5
|
3
|
1
|
4
|
2
|
245.36±4.41
|
240.94
|
47.80
|
|
T21
|
5
|
1
|
5
|
4
|
3
|
2
|
260.3±1.34
|
261.64
|
48.31
|
|
T22
|
5
|
2
|
1
|
5
|
4
|
3
|
293.45±6.3
|
287.15
|
49.35
|
|
T23
|
5
|
3
|
2
|
1
|
5
|
4
|
194.3±18.68
|
212.98
|
45.77
|
|
T24
|
5
|
4
|
3
|
2
|
1
|
5
|
205.36±3.8
|
201.58
|
46.25
|
|
T25
|
5
|
5
|
4
|
3
|
2
|
1
|
280.203±2.2
|
282.39
|
48.95
|
|
Metal Precursor
conc. (M)
|
Reducing/
Capping agent conc. (%)
|
Temperature
(0C)
|
pH
|
Stabilizing agent conc. (%)
|
Reaction time (hr)
|
|
|
L1
|
1.25
|
100
|
60
|
9
|
20
|
5
|
|
L2
|
1
|
80
|
50
|
8
|
15
|
4
|
|
L3
|
0.75
|
60
|
40
|
7
|
10
|
3
|
|
L4
|
0.5
|
40
|
30
|
6
|
5
|
2
|
|
L5
|
0.25
|
20
|
20
|
5
|
0
|
1
|
Table (10): Regression statistics and analysis of variance (ANOVA) for Taguchi experimental design.
|
Coefficients
|
Standard Error
|
t Stat
|
P-value
|
Confidence level (%)
|
|
Intercept
|
321.07
|
6.75
|
47.54
|
2.23113E-20
|
|
|
A
|
5.28
|
0.90
|
5.86
|
1.51738E-05
|
99.98
|
|
B
|
-2.41
|
0.90
|
-2.67
|
0.015746934
|
98.42
|
|
C
|
-11.66
|
0.90
|
-12.92
|
1.52472E-10
|
99.98
|
|
D
|
8.28
|
0.90
|
9.17
|
3.32135E-08
|
99.98
|
|
E
|
-4.27
|
0.90
|
-4.74
|
0.000164547
|
99.98
|
|
F
|
-22.72
|
0.90
|
-25.18
|
1.75391E-15
|
100
|
|
ANOVA
|
|
df
|
SS
|
MS
|
F
|
Significance F
|
|
Regression
|
6
|
38629.40865
|
6438.234775
|
158.1133916
|
1.43084E-14
|
|
Residual
|
18
|
732.9437737
|
40.71909854
|
|
|
|
Total
|
24
|
39362.35242
|
|
|
|
|
Regression Statistics
|
|
Multiple R
|
R Square
|
Adjusted R Square
|
Standard Error
|
Observations
|
|
0.99
|
0.98
|
0.98
|
6.38
|
25
|
Table (11): Bio-application of the fabricated MnO NPs using the endophytic Trichodermavirens strain EG92against phytopathogens in vitro to detect MIC, MFC, and MBC values.
|
Pathogens
|
Fungus-fabricated MnO NPs concentration (µg/ml)
|
|
Zone of inhibition (mm)
|
|
10
|
50
|
90
|
130
|
170
|
210
|
250
|
290
|
330
|
370
|
MFC (µg/ml)
|
MBC (µg/ml)
|
|
Phytopathogenic fungi
|
|
Fusariumsolani
|
9.65±1.34
|
11.51±3.98
|
15.38±4.89
|
16.88±2.89
|
22.50±2.90
|
13.5±1.1
|
18.90±2.89
|
28.75 ± 2.89
|
32.5±1.8
|
21.90±2.90
|
400
|
|
|
Fusariummoniliforme
|
16.59±2.09
|
13.54±2.78
|
13.59±1.29
|
21.09±1.78
|
28.13±2.67
|
16.44 ±2.82
|
25.62±2.78
|
33.19 ± 1.76
|
37.88±3.25
|
33.25±3.56
|
340
|
|
Helminthosporium sp.
|
19.95±1.98
|
18.56±2.54
|
16.41±3.67
|
23.91±1.89
|
31.88±1.04
|
20.06±2.4
|
20.37±1.89
|
35.06 ± 1.34
|
40.38±2.58
|
38.5±2.89
|
420
|
|
Alternaria alternate
|
19.52±2.67
|
13.25±1.67
|
17.81±3.65
|
25.31±0.97
|
33.75±2.09
|
21.13 ±3.05
|
21.25±1.45
|
37.13 ± 3.98
|
42.75±3.96
|
38.5±1.89
|
360
|
|
Aspergillus flavus
|
13.65±0.45
|
16.95±1.89
|
19.69±5.89
|
19.69±2.89
|
26.25±4.23
|
16.88 ±1.51
|
23.75±1.89
|
31.88 ± 2.98
|
36.25±1.63
|
28±2.98
|
380
|
|
Aspergillus niger
|
11.85±2.87
|
16.65±4.98
|
19.41±1.78
|
19.41±2.98
|
25.88±5.89
|
15.56 ± 2.73
|
23.37±1.42
|
30.56 ± 1.25
|
34.875±4.64
|
24.5±1.23
|
400
|
|
Phytophthoraarenaria
|
10.95±1.09
|
12.75±0.45
|
14.34±6.90
|
14.34±2.56
|
19.13±2.89
|
12.44 ±1.78
|
16.62±1.90
|
24.93 ± 2.75
|
28.13±2.97
|
22.75±3.67
|
440
|
|
Phytopathogenic bacteria
|
|
Clavibactermichiganensis
|
8.14±0.05
|
14.30±1.89
|
16.50±0.46
|
20.50±2.90
|
26.91±2.89
|
33.75±1.90
|
30.25±2.90
|
28.63±3.89
|
24.69±2.12
|
20.71±2.89
|
|
220
|
|
Erwiniacarotovora
|
10.73±2.98
|
18.85±2.67
|
21.75±0.56
|
28.13±1.89
|
38.81±3.90
|
42.18±2.56
|
41.5±1.56
|
35.23±2.12
|
35.42±1.93
|
19.69±2.89
|
200
|
|
Acetobacterpasteurianus
|
9.25±2.97
|
16.25±1.89
|
18.75±3.56
|
22.88±3.03
|
28.69±2.54
|
47.81±6.34
|
38.5±3.89
|
31.59±2.89
|
30.99±3.12
|
22.31±2.89
|
240
|
|
Erwiniaamylovora
|
8.51±3.13
|
14.95±2.89
|
17.25±1.09
|
23.75±2.05
|
34.41±1.78
|
50.62±2.34
|
48.75±2.45
|
49.23±3.45
|
48.96±1.34
|
40.28±2.16
|
280
|
|
Erwiniapersicina
|
9.62±2.45
|
16.90±1.34
|
19.50±2.89
|
16.25±1.65
|
29.34±3.56
|
39.37±1.02
|
36.5±1.89
|
22.63±1.23
|
21.92±4.89
|
16.01±5.89
|
200
|