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Production of Bioethanol from Mahua flowers (Madhuca indica) using Saccharomyces cerevisiae with Statistical optimization of Physico-Chemical and Nutritional Factors in Batch Bioreactor by Response
Surface Methodology (RSM) through Submerged Fermentation (SmF)
Prof. C.Ayyanna.B.Tech, Ph.D (I.I.Sc)., M.I.I.Ch.E., F.I.E.
School of of BiotechnologyChemical Engineering Department
Visakhapatnam, Andhra Pradesh, India
ByDr. Dovari Surendra Nadh Benerji
M.Sc, M.Phil, Ph.D
Ph.D awarded in Natural Science Acharya Nagarjuna University
Guntur-522510, India.2014
Research Supervisor
Mahua flower (Madhuca indica)
Microorganisms-Yeast and Bacteria
Fermenter-5L-B-Lite, Sartorious Private Limited, Mumbai, India
Gas Chromatography
AbstractCurrently, the most widely used biofuel produced from the biomass
through fermentation process is Bioethanol, which is now blend of Diesel and Petrol.(Gasohol and Dioseil)
Bioethanol can significantly reduce the accumulation of Green House gases in the Atmosphere.
Bioethanol production from renewable sources have gained the demand for various industrial purposes such as an alternate fuel, solvent in Pharmaceutical industry, Disinfectant, Cleansing agents, Motor fuel, Germicide and as Preservative.
In the present investigations, it was found that Mahua flowers (Madhuca indica) is an alternative agricultural feedstock, which is next to the cane molasses in the world.
Abstract Flowers of Madhuca indica contain High sugar concentrations as well
as mineral ions (Wealth of India, CSIR, 1964,1998, New Delhi). In the present investigation, the Mahua flower contains 73.13 % of
Fermentable sugars, 4.6 mg of Protein, 0.5 % of Fat and Moisture content 17%.
Mahua flower itself has Antibiotic activity. Hence, it was selected as a source of sugars to produce bioethanol through submerged fermentation using yeast and Bacterial strains.
Present studies were carriedout by screening of Saccharomyces cerevisiae-171 MTCC , Kluyveromyces thermotolerance-30 MTCC, S.cerevisiae-3288 NCIM, S.cerevisiae-3190 NCIM, K.marxianus-1389 MTCC, Zymomonas mobilis-92 MTCC, Escherichia coli, S.cerevisiae-463 MTCC.
Research Work (1). Biochemical Analysis of Mahua flower (Madhuca indica)(A). Determination of Moisture Content (AOAC,2000) (B). Estimation of Total Sugars(Anthrone method)(C). Estimation of Reducing Sugars(Dinitro-Salisylic acid reagent)(D). Estimations of Proteins(Lowry’s method)(E). Estimation of Fat (AACC,2000)(2). Screening of Microorganisms for Bioethanol production.(A). Preparation of Nutrient Media(B). Preparation of Nutrient Agar slants(C). Preparation of Nutrient Broth(D). Pure culture of yeast Cells(E). Determination of Number of CFU(F). Selection of Microorganisms for Bioethanol production
through screening process.(G). Total viable cells count(Methylene Blue Reagent)
Biochemical Analysis of Mahua flower (Madhuca Indica)
Moisture content: 17%Total sugars: 731.343 gm/100gmReducing sugars: 18%Protein content: 4.6gmFat:0.5%
Optimization Studies(3). Optimization:- In fermentation process, the suitable Physico-
Chemical and Nutrient conditions favors Growth of Microorganisms and Bioethanol productions were optimized.
pH optimization
The fermentations are carriedout with pH is in the range of 1 to 14, if maximum production of ethanol was obtained at pH 5, which is found to be optimum pH, at which the maximum bioethanol production and maximum yeast cells were obtained. Hence, the process is called “Optimization”.
Standardization of Physico-Chemical and Nutrient conditions
through optimization studies for the preparation of Medium-I on Bioethanol production.
Statistical Optimization Method(4). Statistical optimization studies:- Statistical optimization
was carriedout using Statistica8, Statsoft, USA.(5). Optimization of Physico-Chemical and Nutrient conditions
using Response Surface Methodology.(6). Preparation of Fermentation Medium-II using Statistically
optimized Physico-Chemical and Nutritional conditions to enhance Bioethanol production.
(7). Comparative studies of Medium-I and Medium-II on bioethanol production using potential Microorganism.
(8). Economic Impact of Bioethanol production in India.
Studies on Physico-Chemical and Nutritional Parameters on Bioethanol Production.
Initially, the Physico-Chemical and Nutritional parameters were optimized individually in 5l bioreactor using batch fermentation for bioethanol yields.
Physical Parameters
(1). Substrate Concentration (g.l-1 )(2). Fermentation Time (Hours)(3). Temperature (0C)(4). pH(5). Agitation (RPM)(6). Inoculum Volume (v/v)(7). Inoculum Age (Hours)
Chemicals and Nutritional factors
(1). Inorganic Nitrogen source (A). Ammonium Sulphate (NH4)2 SO4) (B). Ammonium Chloride (NH4 Cl)(2). Copper Chloride (CaCl2) (3). Manganese Chloride (MnCl2.4H2O) (4). Magnesium Chloride (MgCl2.6H2O) (5). Zinc Sulphate (ZnSo4.7H2O)(6). Biotin (8). Proline(9). Glycine(10). Sodium Di-hydrogen Phosphate
(NaH2PO4)
(11). Calcium Chloride (CaCl2) (12). Cobalt Chloride (CoCl2)
(13). Sodium Chloride (NaCl)(14). EDTA(15). Potassium Phosphate (K2HPO4) (16). Oxygen (O2)(17). Ferrous Sulphate (Fe2(So4)3.H2O)(18). Peptone(19). Urea(20). Yeast Extract
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
24000002500000
3400000
3600000
2400000
270000
2300000
210000
Microorganisms Growth
Microorganisms
Num
ber
of c
ells
/ml
24 hours 48 hours 72 hours 92 hours0
5
10
15
20
25
30
35
40
45
50
22.731
43.86542.253
31.7429999999998
Screening of Microorganism
S.cerevisiae-171 MTCC
K.thermotolerance-30 MTCC
S.cerevisiae-3288 NCIM
S.cerevisiae-3190 NCIM
K.marxianus-1389 MTCC
Z.Mobilis-92 MTCC
E.coli
S.cerevisiae-463 MTCC
Fermentation time in hours
Prod
uctio
n of
Bio
etha
nol g
.l-1
40 80 120 160 200 240 280 320 360 400 440 4800
20
40
60
80
100
120
8.413
17.1570000000001
26.129
35.874
45.673
56.193
66.174
77.542
88.1
98.14
65.264
51.531
Effect of Substrate Concentration on Bioethanol Production
Concentration of Substrate (Mahua Flower Extract, MFE) g.L-1
Bioe
than
ol P
rodu
ctio
n g.
l-1
10 20 30 40 500
20
40
60
80
100
120
35.835
59.859
103.15
55.368
44.672
Effect of Temperature on production of Bioethanol % w/v
Temperature (0C)
Bioe
than
ol P
rodu
ctio
n g.
l-1
2 3 4 5 6 7 80
20
40
60
80
100
120
46.856
57.682
84.832
108.69
67.231
55.934
39.693
Effect of pH on Bioethanol production
pH
Prod
uctio
n of
Bio
etha
nol g
.l-1
30 60 90 120 150 180 2100
20
40
60
80
100
120
25.481
36.741
57.446
110.63
47.31
36.481
26.182
Effect of Agitation (RPM) on Bioethanol Production
Agitation (RPM)
Prod
uctio
n of
Bio
etha
nol g
.l-1
4 8 12 16 20 240
20
40
60
80
100
120
77.835
111.19
76.724
65.925
55.472
45.173
Effect of Inoculum volume v/v% on Bioethanol production
Inoculum volume w/v%
Inoculum volume v/v%
Prod
uctio
n of
Bio
etha
nol w
/v%
0.1 0.2 0.3 0.4 0.5
0.600000000000001
0.700000000000001 0.8 0.9 10
20
40
60
80
100
120
36.316
46.458
56.786
67.087
87.353
112.98
98.56
77.56
56.93753.869
30.112
41.923
48.825
59.927
82.628
106.638
88.782
64.853
47.68242.754
Effect of Inorganic Nitrogen source Ammonium sulphate
Ammonium chloride
Inorganic Nitrogen, g.L-1
Prod
uctio
n of
Bio
etha
nol g
.L-1
0.1 0.2 0.3 0.4 0.5
0.600000000000001
0.700000000000001 0.8 0.9 10
20
40
60
80
100
120
56.682
67.753
78.967
89.975
110.189
99.568
78.954
67.684
56.643
45.943
Effect of Copper on Bioethanol production
Copper
Concentration of Copper, Cu+2 , g.l-1
Prod
uctio
n of
Eth
anol
g.l-
1
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
20
40
60
80
100
120
140
24.676
35.748
46.566
54.343
76.099
116.64
88.654
78.578
65.341
54.257
Effect of Manganese on Bioethanol production
Concentration of Manganese g.L-1
Prod
uctio
n of
Bio
etha
nol g
.l-1
0.1 0.2 0.3 0.4 0.5
0.600000000000001
0.700000000000001 0.80
20
40
60
80
100
120
140
66.895
78.539
89.531
118
89.083
68.985
57.375
46.859
Effect of Mg+2 on Bioethanol production
Magnesium
Concentration of Magnesium, g.L-1
Prod
uctio
n of
Eth
anol
, g.L
-1
10 20 30 40 50 60 70 80 900
20
40
60
80
100
120
140
45.653
56.363
67.542
78.682
114.75
85.412
75.473
65.256
54.115
Effect of Zinc on Bioethanol production
Zinc
Zinc mg.L-1
Prod
uctio
n of
Bio
etha
nol g
.L-1
6 12 18 24 30 36 42 48 540
20
40
60
80
100
120
140
55.392
67.302
89.973
115.68110.873
104.537
98.392
88.65285.306
Effect of Biotin on Bioethanol production
Biotin, mg.L-1
Prod
uctio
n of
Bio
etha
nol,
g.l-1
30 60 90 120 150 180 210 240 2700
20
40
60
80
100
120
140
25.968
36.957
57.663
89.391
114.36
75.438
65.875
58.524
47.142
20.741
32.871
51.382
84.812
100.871
112.753
62.561
52.718
42.524
Effect of Proline & Glycine on Bioethanol production
Proline
Glycine
Concentration of Proline and Glycine, mg.L-1
Prod
uctio
n of
Eth
anol
, g.l-
1
1 2 3 4 5 6 7 8 90
20
40
60
80
100
120
140
43.871
54.739
65.431
87.121
119.342
84.135
72.152
64.081
56.004
Effect of Phosphorous on Bioethanol production
Concentration of Phosphorous, g.L-1
Prod
uctio
n B
ioet
hano
l, g.
l-1
1 2 3 4 5 6 7 8 90
20
40
60
80
100
120
140
41.986
52.868
63.866
75.543
117.653
79.858
65.561 63.683
52.659
EDTA effect on Bioethanol Production
EDTA, g.L-1
Prod
uctio
n of
Bio
etha
nol,
g.l-1
0.5 1 1.5 2 2.5 3 3.5 4 4.5 50
20
40
60
80
100
120
140
54.412
66.971
87.099
116.981
78.342
67.097
52.98
45.568
34.56130.102
Effect of Potassium on Bioethanol production
Concentration of Potassium g.L-1
Prod
uctio
n o
f Bio
etha
nol g
.l-1
0
20
40
60
80
100
120
140
34.948
45.473
55.862
76.952
97.642
115.947000000001
85.594
65.287
54.397
43.756
Effect of Calcium on Bioethanol production
Concentration of Calcium, g.L-1
Prod
uctio
n of
Bio
etha
nol g
.l-1
20 40 60 80 100 120 140 160 1800
20
40
60
80
100
120
140
56.303
67.001
87.249
118.635
85.562
67.683
53.742
46.231
35.001
Effect of Cobalt on Bioethanol production
Concentration of Cobalt, mg.L-1
Prod
uctio
n of
Bio
etha
nol,
g.L
-1
0
20
40
60
80
100
120
49.538
63.892
77.728
90.543
102.721
88.782
63.089
43.231
35.00132.782
Effect of Ferrous on Bioethanol production
Concentration of Ferrous, g.L-1
Prod
uctio
n of
Bio
-eth
anol
g.L
-1
0
10
20
30
40
50
60
70
80
90
100
30.7829999999997
61.392
90.53
85.64881.783
74.793
69.69265.891
59.691
50.863
Effect of Oxygen on Bioethanol production
Concentration of Oxygen, mg.L-1
Prod
uctio
n of
Bio
etha
nol g
.L-1
0.2 0.4 0.600000000000001 0.8 0.1 0.12 0.14 0.16 0.18 0.20
10
20
30
40
50
60
70
80
90
100
20.251
36.271
45.957
60.573
93.641
83.673
72.961
63.573
55.7320000000001
42.978
Effect of Sodium Chloride on Bioethanol production
Sodium Chloride, g.L-1
Prod
uctio
n of
Bio
etha
nol,
m g
.L-1
0.5 1 1.5 2 2.5 3 3.5 4 4.5 50
20
40
60
80
100
120
140
50.578
66.628
75.36780.562
92.583
100.63495.976
89.713
75.23970.713
60.142
76.171
85.65790.273
114.735
93.773
82.261
73.363
65.592
52.892
70.783
89.682
118.462
80.863
74.793
65.852
55.756
61.853
55.756
45.972
Effect of Organic Nitrogen on Bioethanol production
Peptone Urea
Yeast Exctract
Organic Nitrogen, g.l-1
Bio
etha
nol p
rodu
ctio
n g.
l-1
Optimal Physical and Chemical Parameters with S.cerevisiae-3190
S.no Parameter Optimal value EtOHProduction
Productivityg.l-1.h-1
EtOH%
1 S.cerevisiae-3190 3.6x106 cells.ml-1/48 hr2 Fermentation time 48 Hours 43.865 0.913 28.613
3 Substrate concentration 400 g.l-1 98.14 2.044 48.013
4 Temperature 30 0C 103.15 2.065 50.4645 pH 5 108.69 2.25 53.1756 Agitation 120 RPM 110.63 2.179 54.1247 Inoculum volume 8 v/v 111.19 2.316 54.3988 Ammonium Sulphate 0.6 g.l-1 112.98 2.353 55.27
10 Copper chloride 0.5 g.l-1 110.189 2.295 53.90811 Manganese chloride 0.06 g.l-1 116.64 2.43 57.06412 Magnesium 0.4 g.l-1 118.00 2.458 57.72913 Zinc sulphate 50 mg.l-1 114.75 2.390 56.13914 Biotin 24 mg.l-1 115.68 2.430 56.59415 Proline 0.150 g.l-1 114.36 2.382 55.94916 EDTA 5 g.l-1 117.653 2.451 57.56017 Phosphorous 5 g.l-1 119.342 2.486 58.38618 Potassium 2.0 g.l-1 116.981 2.437 57.23119 Calcium chloride 0.60 g.l-1 115.947 2.415 56.72520 Cobalt chloride 80 mg.l1 118.635 2.471 58.04021 Ferrous sulphate 0.5 g.l-1 102.721 2.140 50.25422 Oxygen 0.3 mg.l-1 90.530 1.886 44.29023 Sodium chloride 1.0 g.l-1 93.641 1.950 45.81224 Peptone 3 g.l1 100.634 2.090 49.23325 Urea 2.5 g.l-1 114.735 2.390 56.13226 Yeast extract 1.5 g.l-1 118.462 2.467 57.955
Chromatogram of bioethanol and n-butanol
Chromatogram of bioethanol yield with Medium-I
Bioethanol production with Medium-I
24 48 72 960
50
100
150
200
250
300
350
400
255
360
330
299
87.654
150.562 144.537130.638
55
9885 79
Total sugars utilized on bioethanol production,g/l
Bioethanol production,g/l
Yeast viability,%
Fermentation time in Hours
Bio
etha
nol p
rodu
ctio
n, %
Determination of Bioethanol concentrationin fermentation medium
% of Improved Bioethanol= Bioethanol recovery of Medium II – Bioethanol recovery of Medium I
Bioethanol production Medium-I with S.cerevisiae-3190
• Peak area bioethanol = 7054528 • Peak area n-butanol = 11135140 • Wt of standard = 1.3732 mg • Standard volume = 100 ml• Weight of sample = 1.1752 mg• Sample volume = 100 ml• Standard potency of ethyl alcohol = 99.5 • Therefore,• Total content of bioethanol in fermented medium-I
=73.562gm/100ml
Response Surface Methodology• Statistica7 , State Ease Inc., USA (Software Package)• CCD (Central Composite Design) or 23 – Factorial Experimental Designs• Design= 3 variables and 17 Experiments• Number of Centre points (no = 3) where Optimum Concentrations of 3 Variables • N= 2k + 2K + no (where is K=3)• Total Number of Experiments=17• Axial points=2 (with the 1.67332)• Independent Variables= X1, X2 and X3
Coded Values= +1 (Coded value), 0 (Optimum value) and -1 (Low Value)
γi = -β̥+β1x1+ β2x2+ β2x3+ β11 x12+ β22 x2
2+ β33x32+ β12x1x2+ β13x1x3+β23x2x3
β̥ is Offset termβ1, β2 and β3 are Linear effectsβ11, β22 and β33 are Squared effects β12, β13 and β23 are Interaction terms
Analysis Of Variance (ANOVA) table
• Source of Degree of Sum of Mean squares F value Probability˂F• Variation freedom squares • (DF) (SS) (MS) • Due to p-1 SSR MSR/MSE • SSR Regression • (fitted model) • N-p SSE SSE/(N-p)• Residual (error) • N-1 SST• Total
S.no No of ExperimentsUsing CCD
Coded values
(X1) (X2) (X3)
1 1 - 1.00000 - 1.00000 - 1.00000
2 2 - 1.00000 1.00000 1.00000
3 3 1.00000 - 1.00000 1.00000
4 4 1.00000 1.00000 - 1.00000
5 5 0.00000 0.00000 0.00000
6 6 - 1.00000 - 1.00000 1.00000
7 7 - 1.00000 1.00000 - 1.00000
8 8 1.00000 - 1.00000 - 1.00000
9 9 1.00000 1.00000 1.00000
10 10 0.00000 0.00000 0.00000
11 11 - 1.67332 0.00000 0.00000
12 12 1.67332 0.00000 0.00000
13 13 0.00000 - 1.67332 0.00000
14 14 0.00000 1.67332 0.00000
15 15 0.00000 0.00000 - 1.67332
16 16 0.00000 0.00000 1.67332
17 17 0.00000 0.00000 0.00000
S.No Central Composite Design(CCD)
Independent Variables Coded ValuesLow Value
(-1) Center Value
(0)High Value
(+1)
1 Design-ISubstrate Concentration, g.l-1 -1 0 +1Temperature, 0C -1 0 +1
pH -1 0 +1
2 Design-IIInoculum Volume, v/v -1 0 +1
Agitation, RPM -1 0 +1
Inoculum age, Hours -1 0 +1
3 Design-IIIInorganic Nitrogen, g.l-1 -1 0 +1
Copper, g.l-1 -1 0 +1
Manganese, g.l-1 -1 0 +1
4 Design-IVMagnesium, g.l-1 -1 0 +1
Zinc, mg.l-1 -1 0 +1
Vitamins, mg.l-1 -1 0 +1
5 Design-VAmino Acids, g.l-1 -1 0 +1
Phosphorous, g.l-1 -1 0 +1
Metal chelater, g.l-1 -1 0 +1
6 Design-VIPotassium, g.l-1 -1 0 +1
Calcium, g.l-1 -1 0 +1
Cobalt, mg.l-1 -1 0 +1
7 Design-VIIFerrous, g.l-1 -1 0 +1
Oxygen, mg.l-1 -1 0 +1
Sodium Chloride, g.l-1 -1 0 +1
8Design-VIII
Peptone, g.l-1 -1 0 +1
Urea , g.l-1 -1 0 +1
Yeast Extract, g.l-1 -1 0 +1
S.No Central Composite Design(CCD)
Independent Variables Coded ValuesLow Value
(-1) Center Value
(0)High Value
(+1)
1 Design-ISubstrate Concentration, g.l-1 360 400 440
Temperature, 0C 20 30 40
pH 4 5 6
2 Design-IIInoculum Volume, v/v 4 8 12
Agitation, RPM 90 120 150
Inoculum age, Hours 24 48 72
3 Design-IIIAmmonium Sulphate, g.l-1 0.5 0.6 0.7
Copper, g.l-1 0.4 0.5 0.6
Manganese, g.l-1 0.05 0.06 0.07
4 Design-IVMagnesium, g.l-1 0.3 0.4 0.5
Zinc, mg.l-1 40 50 60
Biotin, mg.l-1 18 24 30
5 Design-VProline, g.l-1 0.120 0.150 0.180
Phosphorous, g.l-1 4.0 5.0 6.0
EDTA, g.l-1 4.0 5.0 6.0
6 Design-VIPotassium, g.l-1 1.5 2.0 2.5
Calcium, g.l-1 0.50 0.60 0.70
Cobalt, mg.l-1 60 80 100
7 Design-VIIFerrous, g.l-1 0.4 0.5 0.6
Oxygen, mg.l-1 0.2 0.3 0.4
Sodium Chloride, g.l-1 0.8 1.0 1.2
8Design-VIII
Peptone, g.l-1 2.5 3.0 3.5
Urea , g.l-1 2.0 2.5 3.0
Yeast Extract, g.l-1 1.0 1.5 2.0
CCD of Fermentation Medium-II For Bioethanol ProductionS.No Design Factors
X1 X2 X3
1 Design-I Substrate concentration
Temperature , (0C) pH
2 Design-II Inoculum Volume , (v/v)
Agitation, (RPM) Inoculum Age, Hours
3 Design-III Ammonium Sulphate (NH4)2SO4)
Copper chloride (CuCl2)
Manganese Chloride, (MnCl2.4H2O)
4 Design-IV Magnesium Chloride(MgCl2.6H2O)
Zinc Sulphate(ZnSO4.7H2 O)
Biotin
5 Design-V Proline Sodium Di Hydrogen Phosphorous (NaH2PO4)
EDTA
6 Design-VI Potassium phosphate(K2HPO4)
Calcium Chloride(CaCl2)
Cobalt Chloride(CoCl2)
7 Design-VII Ferrous Sulphate(Fe2(SO4)3.H2O)
Oxygen (O2) Sodium Chloride (NaCl)
8 Design-VIII Peptone Urea Yeast Extract
Optimizations Substrate concentration, Temperature and pH of Design-I on Bioethanol production Using Response Surface Methodology
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Substrate concentration (X1)
409.916 g.l-1
129.4 55.563 2.3660 0.000428Temperature
(X2)31.45 0C
pH (X3) 4.975
Isoresponse surface plot of temperature vs substrate concentration (pH was kept constant at 5) on bioethanol production
Isoresponse counter plot of substrate concentration vs pH (temperature was kept constant at 30 0C) on bioethanol production
Optimizations of Inoculum volume, Agitation and Inoculum Age Design-II on Bioethanol production Using Response Surface Methodology
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Inoculum Volume 9.0003 v/v
121.878 58.185 2.5391 0.440950Agitation 117.28 RPM
Inoculum Age 53.66 Hours
Isoresponse surface plot of inoculum age vs inoculum volume (agitation was kept constant at 120 RPM) on bioethanol production.
Isoresponse counter plot of inoculum age vs inoculum volume (agitation was kept constant at 120 RPM) on bioethanol production.
Optimizations of Ammonium sulphate, Copper and Manganese of Design-III on Bioethanol production Using
Response Surface Methodology
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Ammoniumsulphate
0.629 g.l-1
128.763 61.472 2.6825 0.000155Copper 0.522 g.l-1
Manganese 0.061 g.l-1
Isoresponse surface plot of copper chloride vs ammonium sulphate (manganese was kept constant at 0.06 g.l-1) on bioethanol production.
Isoresponse counter plot of ammonium sulphate vs manganese (copper chloride was kept constant at 0.5 g.l-1) on bioethanol production.
Optimizations of Magnesium, Zinc and Biotin Design-IV on Bioethanol production Using Response Surface Methodology (RSM)
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
Magnesium 0.430
131.281 62.674 2.7350 0.000061Zinc 54.02
Biotin 22.453
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Magnesium 0.430 g.l-1
131.281 62.674 2.7350 0.000061Zinc 54.02 mg.l-1
Biotin 22.453 mg.l-1
Isoresponse surface plot of zinc sulphate vs magnesium chloride (biotin was kept constant at 24 mg.l-1) on bioethanol production.
Isoresponse counter plot of magnesium chloride vs zinc sulphate (biotin was kept constant at 24 mg.l-1) on bioethanol production.
Optimizations of Proline, Phosphorous and EDTA Design-V on Bioethanol production using Response Surface Methodology
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Proline 0.163 g.l-1
129.936 62.032 2.7070 0.000597Sodium Di-Hydrogen Phosphorous
5.385 g.l-1
EDTA 5.197 g.l-1
Isoresponse surface plot of ethylene di-amine tetraacetic acid vs phosphorus (proline was kept constant at 0.150 g.l-1) on bioethanol production.
Isoresponse counter plot of phosphorus vs proline (ethylene di-amine tetraacetic acid was kept constant at 5.0 g.l-1) on bioethanol production.
Optimizations of Potassium phosphate, Calcium chloride and Cobalt chloride of Design-VI on Bioethanol production Using
Response Surface MethodologyVariables Optimum
ConcentrationBioethanol
Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Potassium phosphate
2.170 g.L-1
132.515 63.263 2.7607 0.000914CalciumChloride
0.647g.L-1
Cobalt Chloride 99.486 mg.L-1
Isoresponse surface plot of potassium vs calcium chloride (cobalt chloride was kept constant at 80 mg.l-1) on bioethanol production.
Isoresponse counter plot of calcium chloride vs potassium phosphate (cobalt chloride was kept constant at 80 mg.l-1) on bioethanol production.
Optimizations of Ferrous sulphate, Oxygen and Sodium Chloride of Design-VII on Bioethanol production Using
Response Surface MethodologyVariables Optimum
ConcentrationBioethanol
Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
FerrousSulphate
0.533 g.l-1
125.929 60.119 2.623 0.003863Oxygen 0.330 mg.l-1
Sodium Chloride
1.105 g.l-1
Isoresponse surface plot of oxygen vs ferrous sulphate (sodium chloride was kept constant at 1.0 g.l-1) on bioethanol production.
Isoresponse counter plot of sodium chloride vs ferrous sulphate (oxygen was kept constant at 0.3 mg.l-1) on bioethanol production.
Optimizations of Design-VIII on Bioethanol production Using Response Surface Methodology
Variables Optimum Concentration
Bioethanol Yieldg.l-1
% of Bioethanol
Yield
Productivityg.l.h.-1
P-Value
Peptone 3.038 g.l-1
135.164 64.528 2.8159 0.0665Urea 2.566 g.l-1
Yeast Extract 0.572 g.l-1
Isoresponse surface plot of yeast extract vs peptone (urea was kept constant at 2.5 g.l-1) on bioethanol production.
Isoresponse counter plot of peptone vs yeast extract (urea was kept constant at 2.5 g.l-1) on bioethanol production.
Bioethanol productions using Fermentative Medium-II Designs by Response Surface Methodology
MediumDesign
Bioethanol Yield g.L-1
% of Bioethanol
Productivityg.l.h-1
P-Value
Design-I 129.4 55.563 2.3660 0.000428
Design-II 121.878 58.185 2.5391 0.440950
Design-III 128.763 61.472 2.6825 0.000155
Design-IV 131.281 62.674 2.7350 0.000061
Design-V 129.936 62.032 2.7070 0.000597
Design-VI 132.515 63.263 2.7607 0.000914
Design-VII 125.929 60.119 2.623 0.003863
Design-VIII 135.164 64.528 2.8159 0.0665
Statistical optimum fermentative conditions of Medium-IIS.No CCD Factors Unit Statistical optimum fermentative conditions
1 Design-I Substrate conc’nTemperaturepH
g/l0CpH
409.9314.9
2 Design-II Inoculum volumeAgitationInoculum age
v/vRPMHours
9.00311753.6
3 Design-III Ammonium sulphateCopperManganese
g/lg/lg/l
0.6290.5220.061
4 Design-IV MagnesiumZincBiotin
g/lmg/lmg/l
0.43054.02122.453
5 Design-V ProlinePhosphorusEDTA
g/lg/lg/l
0.1635.3855.197
6 Design-VI PotassiumCalciumCobalt
g/lg/lmg/l
2.3400.06499.43
7 Design-VII
FerrousOxygenNaCl
g/lmg/lg/l
0.5330.3301.105
8 Design-VII
PeptoneUreaYeast Extract
g/lg/lg/l
3.0382.5660.572
Chromatogram of Bioethanol with medium-II
Bioethanol production with Medium-II
24 hours 48hours 72 hours 96 hours0
50
100
150
200
250
300
350
400
450
260
408
350
320
89.875
195.284
173.561
150.863
75
9786 81
total fermentable sugars uti-lized,g/l
bioethanol concentration,g/l
total yeast viability,%
Fermentation time (hours)
Bio
etha
nol p
rodu
ctio
n, g
/l
Bioethanol Production Using Medium-II
Peak Area of Bioethanol = 9123399 Peak Area of n-Butanol = 11135140 Wt of standard = 1.3732 mgStandard Volume = 100 mlWt. of Sample = 1.2008 mgSample Volume = 100 mlStandard Potency = 99.5 %Therefore,Percentage of bioethanol production of Medium-II = 93.229 %
Economic importance of bioethanol
Bioethanol:3,48,303 tonns/1 million tonns of Madhuca indica.
When bioethanol used as blend, thus replaces 3,48,303 tonns of petrol.
Gasohol can reduce Green House Gases.
