Optimization of Biodiesel preparation by enzymatic transesterification using Mahua oil and

study on utilization of glycerol into Bioethanolby

S. Mozhiarasu1* & B.Aswin Kumar*

*Madha Engineering College1Research Associate, Bio Globe

Scientific Park

To study about different trans-esterification

methods To optimize the reaction conditions for

enzymatic method To study ethanol production using waste

glycerol

OBJECTIVE

Biodiesel consists of Fatty acid alkyl esters It can be produced by transesterification of

Triglycerides and alcohol in the presence of catalyst.

Introduction

Major Methods of Transesterification

Alkali mediated Method

Acid mediated method

Enzymatic Method

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

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.

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

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.

HYDROLASE ACTIVITY

ESTERASE ACTIVITY

Factors influencing production of biodiesel

Temperature pH

Stirring MeOH :Oil Buffer/Water concentration Enzyme concentration Stepwise addtion

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 ASSAYEstimation 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

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

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 %

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 bufferReaction Time: 7 hrsRPM : 100 RPM in Rotary

Shaker

Effect Of Temperature

Effect Of pH of Buffer

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

Taguchi L16 ArrayRun 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

Nominal is Best approach:

S/Nt = -10 log (yi2/ 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

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

4321

32

30

28

26

244321 4321

4321

32

30

28

26

244321

Oil: MeOHM

ean o

f SN r

atios

Buffer Amount Enzyme Amount

Mixing Rate Stepwise addition Rate of MeOH

Signal-to-noise: Nominal is best (10*Log10(Ybar**2/s**2))

4321

60

50

40

30

20

4321 4321

4321

60

50

40

30

20

4321

Oil: MeOH

Mean o

f M

eans

Buffer Amount Enzyme Amount

Mixing Rate Stepwise addition Rate of MeOH

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

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

MeOH3 163 54 0.07 0.973

Total 15 8981

ANOVA table for all the factors

OPTIMIZED VALUE

Temperature (oC) ` - 35pH of Buffer - 7.0Oil:MeOH (mol:mol) - 1:1.5Buffer amount (w/w) - 15%Enzyme amount (w/w) - 0.25%Mixing Rate (RPM) - 100Stepwise 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

Still Lot To Travel

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