Field of the Invention
The present invention relates to a process for the production of bio-diesel. More particularly, the
invention is concerned about a process comprising enzymatic transesterification of jatropha oil
by means of improved stabilization of enzyme for preparation of bio-diesel. The said bio-diesel
can act as a substitute for, or an additive to diesel-fuel used as automotive or other vehicular
fuel and lubricants.
BACKGROUND AND THE PRIOR ART
Significant quantities of esters such as triglycerides and free fatty acids are available from
inexpensive feedstocks such as animal fats, vegetable oils, rendered fats, restaurant grease
and waster industrial frying oil. An enzyme catalyzed transesterification reaction is carried out
for the production of biodiesel from the inexpensive feedstocks.
US 5713965 describe a method which utilizes lipases to transesterify triglyceride containing
substances and to esterify free fatty acids to alkyl esters using short chain alcohols. However,
the method involves lipase from Mucor meihei and Candida antartica. The present invention
discloses the use of lipase from Enterobacter aerogenes, which has been used for the first
time for such transesterification studies.
US 5578090 discloses a fuel additive composition including fatty acid alkyl esters and glyceryl
ethers, and an alternative fuel composition that includes the fuel additive composition, wherein
the fuel additive composition is prepared by esterifying free fatty acids and etherifying glycerol
with one or more olefins in the presence of an acid catalyst. It was not known that that
transesterification reaction can be carried out in the presence of an enzyme with improved
stabilization of the enzyme.
US 6855838 relates to a method for producing fatty acid alkyl esters, involving esterifying a
material containing free fatty acids (FFA) with an alcohol and an inorganic acid catalyst to form
a product containing fatty acid alkyl esters. It was not known that that transesterification reaction
can be carried out in the presence of an enzyme with improved stabilization of the enzyme.
US 6822105 provide a method of processing free fatty acids from a vegetable or animal oil
source into alkyl esters in which the salt and aqueous waste is reduced or eliminated. The
method is performed in the presence of a catalyst wherein the catalyst is selected from the
2

group consisting of organotin compounds, organo titanium compounds, alkali acetates, earth
alkali acetates, Lewis acids, alkali carbonates, earth alkali carbonates, and combinations
thereof. It was not known that that transesterification reaction can be carried out in the presence
of an enzyme with improved stabilization of the enzyme.
US 6768015 describe method for making alkyl esters, or methylester specifically, such as
biodiesel, from an oil source is described. The method involves simultaneously reacting the free
fatty acids and glycerides of the oil source with methanol, under pressure up to 500 psia, into
fatty acid alkyl esters. The conversion is catalyzed by an acid at temperatures between about
80° C to about 200° C. It was not known that that transesterification reaction can be carried out
in the presence of an enzyme with improved stabilization of the enzyme.
US6712867 describes a process for the esterification of a triglyceride in which the base catalyst
is selected from sodium hydroxide and potassium hydroxide. It was not known that that
transesterification reaction can be carried out in the presence of an enzyme with improved
stabilization of the enzyme.
US 6642399 describe a process for the esterification of a mixture of fatty acids and triglycerides.
In a first step, an acid catalyst for the esterification of the fatty acids is added. After a period of
time, the acid catalyst is neutralized and a base catalyst for the transesterification of
triglycerides is added. It was not known that that transesterification reaction can be carried out
in the presence of an enzyme with improved stabilization of the enzyme.
US 6399800 describes a method for producing fatty acid alkyl esters from a feedstock, involving
esterifying the dried saponified feedstock with an alcohol in the presence of an inorganic acid
catalyst to form fatty acid alkyl esters. It was not known that that transesterification reaction can
be carried out in the presence of an enzyme with improved stabilization of the enzyme.
US6398707 describes a technique for enhancing the activity of an immobilized lipase, and a
technique for regenerating a deactivated immobilized lipase, in which an alcohol with a carbon
atom number not less than three is used to swell and/or clean said immobilized lipase. Said
immobilized lipase is particularly useful in a method of preparing biodiesel by transesterification
of triglycerides and a lower alcohol. It was not known that that transesterification reaction can be
carried out in the presence of an enzyme with improved stabilization of the enzyme.
US 6015440 describes a process for producing biodiesel fuel with reduced viscosity and a cloud
point below 32° F. Triglycerides are reacted in a liquid phase reaction with methanol and a
3

homogeneous basic catalyst. The glycerol phase is passed through a strong cationic ion
exchanger to remove anions, resulting in a neutral product which is flashed to remove methanol
and which is reacted with isobutylene in the presence of a strong acid catalyst to produce
glycerol ethers. The glycerol ethers are then added back to the upper located methyl ethyl ester
phase to provide an improved biodiesel fuel. It was not known that that transesterification
reaction can be carried out in the presence of an enzyme with improved stabilization of the
enzyme.
WO 00/05327 provides a single-phase process for producing alkyl esters useful as biofuels and
lubricants by the reactions of triglyceride esters and free fatty acids. This invention teaches a
process where the reactants enter a reactor, whether batch or continuous, dissolved in a critical
fluid. The critical fluid provides a single-phase medium in which diffusion of the reactants into
different liquid phases is eliminated, and mass transfer limitations are essentially eliminated
thereby increasing the overall reaction rate. It was not known that that transesterification
reaction can be carried out in the presence of an enzyme with improved stabilization of the
enzyme.
WO/2004/048311 describes a method for production of alkyl esters involving acid catalysis. It
was not known that that transesterification reaction can be carried out in the presence of an
enzyme with improved stabilization of the enzyme.
WO/2005/021697 describes a method by which high-purity fatty acid alkyl esters and/or
glycerine can be produced advantageously from the energy viewpoint while reducing the energy
consumption of the production system in the production as well as a fatty acid alkyl esters-
containing composition useful for a various way as a biodiesel fuel or a surfactant intermediate.
The method uses an insoluble solid catalyst favorably which results in increased fatty acid alkyl
esters content in the ester phase, hence in reductions in purification cost, improvements in
isolated yield, and omission of the steps of neutralization and acid precipitation. It was not
known that that transesterification reaction can be carried out in the presence of an enzyme with
improved stabilization of the enzyme.
WO/2005/016560 relates to a method and apparatus for producing fatty acid alkyl ester wherein
the method uses solid catalyst for the transesterification reaction. It was not known that that
transesterification reaction can be carried out in the presence of an enzyme with improved
stabilization of the enzyme.
4

Thus there is a need to provide for a process comprising enzymatic transesterification of
jatropha oil by means of improved stabilization of enzyme for preparation of bio-diesel.
The present inventors have found that while preparing bio diesel by means of transesterification
of jatropha oil, improvements in the fuel properties of the bio diesel are obtained when the
jatropha oil transesterification is carried in presence of a catalytic enzyme in combination with a
specific solvent. It has also been found that the process of preparing bio-diesel of the present
invention is less energy consuming as there is no requirement of external source of
heat/energy.
OBJECTS OF THE INVENTION
Accordingly, one object of the present invention is to provide a process for obtaining bio diesel
with improved fuel properties by transesterification of jatropha oil.
Another object of the present invention is to provide enzymatic catalyzation of jatropha oil.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a process for preparation of
bio diesel from jatropha oil, said process comprising :
i) adding an alcohol to the jatropha oil;
ii) adding solvent to said alcohol and oil mixture;
iii) adding lipase to said mixture of jatropha oil, alcohol and solvent
wherein said solvent minimizes interaction of chemicals with lipase thereby resulting
in minimum inhibition to active site of the lipase.
DESCRIPTION OF THE INVENTION
The present invention is a development of an environment friendly process for the production of
biodiesel from jatropha oil wherein there is no application of acid or base. The present invention
relates to a process of enzymatic transesterification of jatropha oil using lipase from
Enterobactor aerogenes (IABR-0785). The application of the lipase from Enterobactor
aerogenes has been used for the first time for such transesterification reaction.
Conventionally, in chemical transesterification process degummiing is one of the techniques,
which are carried out to take care of the phospholipids, present in oil gum, which is not required
for the present invention process. The presence of free fatty acids in chemical transesterification
process interferes resulting in saponification which can be overcome by the present invention.
5

Methanol and the by-product glycerol inhibit lipase activity during bio-diesel synthesis from
vegetable oil. In the present invention t-butanol solvent is used in which both methanol as well
as glycerol is soluble thus minimizing the interaction of the chemicals with the enzyme resulting
in minimum inhibition to the active site of the enzyme.
The present invention describes a process for the synthesis of biodiesel by transesterification of
jatropha oil using lipase from Enterobactor aerogenes (IABR-0785) and was found to give
appreciable yield. In the present invention reaction was carried out with suitable methanol:
jatropha oil ratio in the presence of enzyme. The range of ratio of the methanol to jatropha oil
varies from 2:1 to 4:1.
According to a preferred featured the present invention uses t-butanol as solvent. The process
generates glycerol as a by-product of the transesterification reaction. As described above both
methanol and glycerol dissolves in t- butanol, which reduces the interaction of the chemicals
with the enzyme thereby improving the enzyme stability. Further, the solvent is separated from
the mixture by way of distillation. As the final step of the process the by-product glycerol is
separated and the final product is obtained. Synthesis of fatty acid methyl ester was monitored
by gas chromatography.
It is a preferred aspect of the present invention that the enzyme presently used for
transesterification reaction is stable in organic solvent rather the stability of the enzyme is
increased in the solvent as used in the present invention.
Table 1 provides a comparative study data of improved fuel properties of jatropha biodiesel from
crude jatropha oil.
Table 1

Property Jatropha oil Jatropha biodiesel
Viscosity (mm2/s) 24.5 8.2
Flash point (°C) 210 195
Pour point (°C) 10 5
Caloric value (MJ/Kg) 36.2 36.5
Thus it is found that jatropha biodiesel has improved fuel properties than jatropha oil.
6

We claim
1. A process for preparation of bio diesel from jatropha oil, said process comprising :
(i) adding an alcohol to the jatropha oil;
(ii) adding solvent to said alcohol and oil mixture;
(iii) adding lipase to said mixture of jatropha oil, alcohol and solvent
wherein said solvent minimizes interaction of chemicals with lipase thereby
resulting in minimum inhibition to active site of the lipase.
7
2. Process as claimed in claim 1, wherein the alcohol is methanol.
3. Process as claimed in claims 1 and 2, wherein the ratio of methanol to jatropha oil
ranges from 2:1 to 4:1.
4. Process as claimed in claim 1, wherein said solvent is t-butanol.
5. Process as claimed in any of the preceding claims, wherein said jatropha oil is
transesterified to a fatty acid ester.
6. Process as claimed in any of the preceding claims further comprising step of
distillation of said fatty acid ester so as to remove remaining solvent.
7. Process as claimed in anyone of the preceding claims wherein transesterification is
carried out at room temperature.

The present invention relates to a process for preparation of bio diesel from jatropha oil.
The process comprises adding an alcohol to the jatropha oil, adding solvent to said
alcohol and oil mixture, adding lipase to said mixture of jatropha oil, alcohol and solvent
wherein said solvent minimizes interaction of chemicals with lipase thereby resulting in
minimum inhibition to active site of the lipase.