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 Biotechnology Chemical Engineering Department Visakhapatnam, Andhra Pradesh, India By Dr. 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
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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)
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.
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)
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
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
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.