Our project on Biodiesel production optimization from Mahua Oil

1. *Madha Engineering College
1Research Associate, Bio Globe Scientific Park

2. 
 To study about different trans-esterification methods
 To optimize the reaction conditions for enzymatic
method
 To study ethanol production using waste glycerol
OBJECTIVE

3. 
 Biodiesel consists of Fatty acid alkyl esters
 It can be produced by transesterification of
Triglycerides and alcohol in the presence of catalyst.
Introduction

4. 
Major Methods of
Transesterification
Alkali mediated
Method
Acid mediated
method
Enzymatic
Method

5. 
1. Mahua oil
o Next to sugarcane, Mahua flowers or fruit
pulp are the most important source of raw
materials for fermentation and production of
alcohol and vinegar.
o Mahua oil seed cake can be used as manure
and as substrate for Lipase production
o Seeds containing 20 to 50% fatty oil
Low Cost Feed Stock

6. Take 1 ml of the reaction mixture
at each interval and 10 ml of
neutralized Iso propyl alcohol is
added
2-3 drops of phenolphthalein
solution is added
Then titrated against 0.1N NaOH
solution
Appearance of Pale pink color is
the end point
Lipase AssayHydrolysis Activity :
µmole oleic acid /ml subsample = ([(ml
NaOH for sample − ml NaOH for blank) × N
× 1000]/quantity of sample) /282.5
One unit (IU) of lipase (hydrolysis) was
defined as the amount of enzyme releasing
one μmol fatty acid equivalent per minute
under the assay conditions.
Esterification Activity:
µmole oleic acid /ml subsample = ([(ml
NaOH for blank− ml NaOH for sample) ×
N × 1000]/quantity of sample) /282.5
One unit (IU) of lipase( esterfication) was
defined as the amount of enzyme
consumed one μmol fatty acid equivalent
per minute under the assay conditions.

7. 
 Take 50 ml conical flask, in which add 5 g of Mahua
Oil and add 40 ml of water
 Then the reaction started by adding 5ml of
phosphate buffer(pH 8.0) containing enzyme of
concentration (0.6 mg/ml)
 The reaction mixture is kept at 100 RPM in a rotary
shaker
 Then for every 5 minutes the reacting mixture is
assayed titer metrically.
Hydrolase activity

8. 
Esterase Activity
 Take 50 ml of flat bottomed flask with stopper, in which
add (1:1 mol/mol) of Methanol:lauric acid,i.e., (1g of
MeOH : 9.32g of Lauric acid). Then add 35 ml of Hexane.
 Then heated at 43oC at 200 RPM until the fatty acid gets
melted down.
 Then the reaction started by adding 5ml of phosphate
buffer containing enzyme of concentration (0.6 mg/ml)
 Then for every 20 minutes the reacting mixture is assayed
titer metrically.

9. HYDROLASE ACTIVITY
ESTERASE ACTIVITY

10. 
Factors influencing production of
biodiesel
 Temperature
 pH
 Stirring
 MeOH :Oil
 Buffer/Water concentration
 Enzyme concentration
 Stepwise addtion

11. 
1g oil+ 0.5N NaOH
at 60oC for 30
minutes
Neutralized with
2mL of 0.5 N HCl
then 5mL of Diethyl
ether was added
Bottom layer
diluted two fold
with ethanol water
CONVERSION ASSAY
Estimation of glycerides present in the form of unconverted oil in the
prepared ester. Total glycerides in the oil and unconverted was done in terms
of glycerol.
Modified method described in P.K.Gupta et. al 2007

12. 
1ml of sample + 1.2 ml 10mM sodium periodate prepared with 10 ml equal
volume of 4 M ammonium acetate and 1.6 M acetic acid , Shaken for 30 seconds
Then add 1.2 ml of 0.2 M Acetyl acetone solution prepared with prepared with
10 ml equal volume of 4 M ammonium acetate and 1.6 M acetic acid and
heated at 70oC for minute
Then OD was measured at 410 nm
Glycerol Assay
As described in bondiolliet.al

13. 
Glycerol equivalent converted = Total glycerol present in oil –
(Total glycerol present in saponified biodiesel sample – Glycerol
present in biodiesel sample (before saponified)
Theoretical biodiesel yield (T) = 3 x Moles of glycerol present in oil
Actual biodiesel (A) = 3 x Moles of Glycerol equivalent converted –
moles of FFA present in the diluted sample.
%Yield = (A / T) x 100
Yield %

14. 
Enzymatic
Transesterification
 Reaction mixture: Reaction mixture consist of
Oil-MeOH ratio : 50 g : 6.4 g ( 1:1 mol/mol)
Enzyme : 0.5% (w/w) mixed with 5 ml
of phosphate buffer
Reaction Time : 7 hrs
RPM : 100 RPM in Rotary Shaker

15. 
Effect Of Temperature

16. 
Effect Of pH of Buffer

17. Variable analysed by Taguchi method
Factor Level 1 Level 2 Level 3 Level 4
Oil : MeOH(mol:mol) 1:1 1:1.5 1:2 1:2.5
Buffer(w/w) 5% 10% 15% 20%
Enzyme Concentration(w/w) 0.25% 0.5% 0.75% 1%
Mixing rate (RPM) 25 50 75 100
Stepwise addition(mol/hr) 0.5 0.625 0.75 0.875

18. Taguchi L16 Array
Run no Oil: MeOH Buffer Enzyme RPM Stepwise addition
1 1 1 1 1 1
2 1 2 2 2 2
3 1 3 3 3 3
4 1 4 4 4 4
5 2 1 2 3 4
6 2 2 1 4 3
7 2 3 4 1 2
8 2 4 3 2 1
9 3 1 3 4 2
10 3 2 4 3 1
11 3 3 1 2 4
12 3 4 2 1 3
13 4 1 4 2 3
14 4 2 3 1 4
15 4 3 2 4 1
16 4 4 1 3 2

19. 
Nominal is Best approach:
S/Nt = -10 log (yi
2/ Si
2)
y = Performance measure.
i = Experiment number
Si = Standard deviation for experiment.
∆= Maximum-Minimum
Optimum S/N ratio = Average S/N + ∑ (Factor effects of all factors).
S/N Ratio

20. Run No Factors Yield % S/N Ratio
Oil:MeOH Buffer % Enzyme % RPM Stepwise
addition
1. 1:1 5 0.25 25 0.5 18.88 23.4150
2. 1:1 10 0.5 50 0.625 34.88 26.3702
3. 1:1 15 0.75 75 0.75 73.95 34.3278
4. 1:1 20 1 100 0.875 70.62 34.5848
5. 1:1.5 5 0.5 75 0.875 27.72 27.3281
6. 1:1.5 10 0.25 100 0.75 86.80 38.0741
7. 1:1.5 15 1 25 0.625 37.60 29.8802
8. 1:1.5 20 0.75 50 0.5 61.48 34.3221
9. 1:2 5 0.75 100 0.625 29.65 25.7016
10. 1:2 10 1 75 0.5 71.28 35.0764
11. 1:2 15 0.25 50 0.875 66.05 35.4496
12. 1:2 20 0.5 25 0.75 16.38 24.9106
13. 1:2.5 5 1 50 0.75 24.63 28.5860
14. 1:2.5 10 0.75 25 0.875 11.27 20.8652
15. 1:2.5 15 0.5 100 0.5 44.03 30.4517
16. 1:2.5 20 0.25 75 0.625 69.30 34.6736
EXPERIMENTAL RESULTS

21. 4321
32
30
28
26
24
4321 4321
4321
32
30
28
26
24
4321
Oil: MeOH
MeanofSNratios
Buffer Amount Enzyme Amount
Mixing Rate Stepwise addition Rate of MeOH
Signal-to-noise: Nominal is best (10*Log10(Ybar**2/s**2))

22. 4321
60
50
40
30
20
4321 4321
4321
60
50
40
30
20
4321
Oil: MeOH
MeanofMeans
Buffer Amount Enzyme Amount
Mixing Rate Stepwise addition Rate of MeOH

23. Level Oil: MeOH Buffer Conc. Enzyme Conc. RPM Stepwise addition rate of MeOH
1 29.67 26.26 32.90 24.77 30.82
2 32.40 30.10 27.27 31.18 29.16
3 30.28 32.53 28.80 32.85 31.47
4 28.64 32.12 32.03 32.20 29.56
Delta 3.76 6.27 5.64 8.08 2.32
Rank 4 2 3 1 5
Response Table for S/N Ratios

24. Source DF SS MS F P
Oil: MeOH 3 584 195 0.28 0.840
Buffer Conc. 3 2470 823 1.52 0.260
Enzyme Conc. 3 1918 639 1.09 0.392
RPM 3 3846 1282 3.00 0.073
Stepwise addition rate of MeOH 3 163 54 0.07 0.973
Total 15 8981
ANOVA table for all the factors

25. OPTIMIZED VALUE
Temperature (oC) ` - 35
pH of Buffer - 7.0
Oil:MeOH (mol:mol) - 1:1.5
Buffer amount (w/w) - 15%
Enzyme amount (w/w) - 0.25%
Mixing Rate (RPM) - 100
Stepwise addition (mol/h) - 0.5
optimum values were tested and yield% were calculated and it was
found that 89.32 ± 2.4% yield was obtained after 7 hrs of reaction
time

26. Still Lot To Travel
THANK YOU