Optimization of Biodiesel preparation by enzymatic transesterification using Mahua oil and study on utilization of glycerol into Bioethanol by S. Mozhiarasu 1* & B.Aswin Kumar* *Madha Engineering College 1 Research Associate, Bio Globe Scientific Park
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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
([(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.
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