US007550278B2

(12) United States Patent (10) Patent No.: US 7,550,278 B2

(54) PROCESS FOR PRODUCING BIODIESEL JP 11263750 9, 1999

(75) Inventors: Wei Du, Beijing (CN): Dehua Liu, Soumanou, M.M. et al., Improvement in Lipase-catalyzed synthesis
Beijing (CN); Lilin Li, Beijing (CN) ffatty acid methyl esters from Sunflower oil, Jul. 16, 2003, Enzyme
and Microbial Technology, vol. 33, pp. 97-103.*
(73) Assignee: Tsinghua University, Beijing (CN) JP 2002-233393, National Research Inst of Brewing, English trans
lation, Aug. 20, 2002.*
(*) Notice: Subject to any disclaimer, the term of this WO 03004591, Siegfried et al., English translation, Jan. 16, 2003.*
patent is extended or adjusted under 35 Nie K. Wang F., Tan T. "Biodiesel Production by Immobilized
Lipase':Modern Chemical Industry 23:9 (2003) pp. 35-38.
U.S.C. 154(b) by 229 days. Shimada; Y.; Watanabe, Y.; Sugihara, A.; Tominaga, Y.: “Enzymatic
alcoholysis for biodiesel fuel production and application of the reac
(21) Appl. No.: 10/597,123 tion to oil processing: Journal of Molecular Catalysis B: Enzymatic
(2002) vol. 17, No. 32 pp. 133-142.
(22) PCT Filed: Nov. 29, 2004 Nelson, L.; Foglia, T. and Marmer, W.:"Lipase-Catalysed Production
of Biodiesel': Journal of the American Oil Chemists' Society, AOCS
(86). PCT No.: PCT/CN2004/001372 Press, Champaign, IL; vol. 73, No. 9, Sep. 1, 1996; pp. 1191-1 195;
XP000626753, ISSN: 003-021X.
S371 (c)(1), Iso, M., Chen, B., Eguchi, M., Kudo, T. and Shrestha, S.; “Production
(2), (4) Date: Dec. 1, 2006 of biodiesel fuel from triglycerides and alcohol using immobilized
lipase'; Journal of Molecular Catalysis B: Enzymatic, Elsevier,
(87) PCT Pub. No.: WO2005/075615 Amsterdam, NL, vol. 16, No. 1 Nov. 20, 2001, pp. 53-58,
XP002417337; ISSN: 1381-1177.
PCT Pub. Date: Aug. 18, 2005 * cited by examiner
(65) Prior Publication Data Primary Examiner Porfirio Nazario Gonzalez
Assistant Examiner Yate" K Cutliff
US 2008/0038804 A1 Feb. 14, 2008
(74) Attorney, Agent, or Firm Gottlieb, Rackman &
(30) Foreign Application Priority Data Reisman, P.C.
Jan. 16, 2004 (CN) ........................ 2004. 1 OOOO697 (57) ABSTRACT
(51) Int. Cl. The present invention provides a new process for producing
CI2P 7/62 (2006.01) biodiesel from renewable oil under lipase catalysis in an
CIIC I/00 (2006.01) organic medium reaction system. In this process, a short chain
CI2P 7/64 (2006.01) alcohol ROH is used as an acyl acceptor, a relatively hydro
CI2P 7/00 (2006.01) philic organic solvent having no negative effect on the reac
(52) U.S. Cl. ......................... 435/135; 435/41; 435/132: tivity of lipase is used as the reaction medium, and a renew
435/134: 554/169 able oil raw material is catalyzed by a lipase to synthesize
(58) Field of Classification Search ................... 435/41 biodiesel through a transesterification reaction. In the process
435/132, 134, 135; 554/169 of the present invention, the renewable oil raw material can be
See application file for complete search history. almost completely converted into biodiesel and a byproduct
glycerin, and the yield of the biodiesel is 94% or more. This
(56) References Cited new type organic medium reaction system promotes the solu
U.S. PATENT DOCUMENTS bility of the short chain alcohol in the renewable oil raw
material, and dissolves part of the byproduct glycerin. Con
6,398,707 B1 * 6/2002 Wu et al. .................... 554,169 sequently, the reaction time is shortened, the productivity of
FOREIGN PATENT DOCUMENTS
biodiesel is increased and the reactivity and lifetime of the
lipase is improved in the process of the present invention.
DE 102176O7 10, 2002
DE WOO3OO4591 ck 1, 2003 11 Claims, No Drawings
 US 7,550,278 B2
1. 2
PROCESS FOR PRODUCING BODESEL hydrophilic glycerin readily adheres to the inner pores and the
FROM RENEWABLE OL UNDER LIPASE outer surface of the immobilized enzyme forming a "shield'
CATALYSIS IN AN ORGANIC MEDIUM on the active sites of the enzyme, and severely affecting the
REACTION SYSTEM reactivity of the enzyme. Furthermore, the yield of biodiesel
synthesized by the enzyme process is relatively low com
TECHNICAL FIELD pared with that by the conventional chemical process. To
Solve above-mentioned problems in a biological enzyme pro
The present invention relates to the field of bio-fuel syn cess, some scholars have tried to produce biodiesel in organic
thesis, and more particularly, relates to a new process for Solvent reaction system. It is reported in an article entitled
producing biodiesel from renewable oil under lipase catalysis 10 Enzymatic alcoholysis for biodiesel fuel production and
in an organic medium reaction system. application of the reaction to oil processing (Journal of
Molecular Catalysis B: Enzymatic, 2002, 17:133-142) writ
BACKGROUND ten by Shimada Yuji et all that, some relatively strong hydro
phobic organic solvents, such as hexane, cyclohexane and
As a promising material of renewable oil industry, biodie 15 petroleum ether can be used as reaction medium. These
sel is along chainfatty acid esterproduced from renewable oil hydrophobic solvents can dissolve renewable oil very well,
through transesterification reaction. Biodiesel is a novel, pol thus promoting the reaction to Some extent. However,
lutionless and recoverable energy source. The combustion because these highly hydrophobic solvents can not dissolve
performance of biodiesel is comparable with the conventional lower carbon alcohol. Such as methanol, as well as the
petroleum-based diesel, while after combustion, the content byproduct glycerin effectively, the reactivity and lifetime of
of harmful Substances in the exhaust gas decreases approxi the enzyme, and the yield of biodiesel can not be improved
mately by 50% as compared with the conventional petro notably.
leum-based diesel. By far, wide attentions have been drawn to
the studies and applications regarding biodiesel. SUMMARY OF THE INVENTION
Currently, biodiesel is manufactured mainly through 25
chemical methods. Particularly, vegetable oils and/or animal Therefore, the object of the present invention is to provide
fats are used as a source of long chain fatty acids and a a new process for producing biodiesel from renewable oil raw
transesterification reaction between the long chain fatty acids material under lipase catalysis in an organic medium reaction
and some short chain alcohols, such as methanol or ethanol, is system. In this process, a short chain alcohol ROH is used as
carried out in the presence of an acid or base catalyst, and 30 an acyl acceptor, a relatively hydrophilic organic solvent hav
short chain ester of the fatty acids are obtained. However, ing no negative effect on the reactivity of lipase is used as the
some inevitable disadvantages exist in chemical methods as reaction medium, and a renewable oil raw material is cata
follows: lyzed by a lipase to synthesize biodiesel through a transes
(1). Free fatty acid and water contained in the renewable oil terification reaction. The process of the present invention is
raw material severely spoil the reaction; 35 characterized in that: a short chain alcohol and arenewable oil
(2). Emulsion is undesirably formed due to the poor solu raw material having an alcohol/renewable oil molar ratio of
bility of alcohol in renewable oils, and the subsequent treat 3:1 to 6:1, together with 20 to 200% by volume of an organic
ment steps are complicated; solvent based on the volume of the renewable oil, and 2 to
(3). As required by the process, the amount of short chain 30% by weight of a lipase based on the weight of the renew
alcohol used is much more than the reaction molar ratio, and 40 able oil are added into an enzyme reactor and mixed evenly,
the evaporation/reflux of the excess short chain alcohol leads and the mixture is heated to 20 to 60°C. to react for 4-24 hours
to increased energy consumption. to convert the renewable oil raw material into biodiesel and a
In contrast, synthesizing biodiesel through a biological byproduct glycerin.
enzyme method has the following advantages: mild reaction
conditions, non-toxic emissions, and enzyme-catalyzed reac 45 BEST MODE OF THE INVENTION
tions are not affected by the free fatty acid and small amount
of water contained in the renewable oil raw materials. There In the process according to the present invention, said
fore, bio-methods are consistent with the requirement of relatively hydrophilic organic solvent is preferably selected
developing Green Chemistry, and thus have attracted more from the group consisting of tert-butanol and short chain fatty
and more attention. 50 acid ester RCOOR', wherein R and R' are independently an
However, when compared with the chemical process, there alkyl group having 1-4 carbon atoms.
exist some problems in the biological enzyme process. In an Said lipase is preferably a microorganism lipase, and is
article entitled Enzymatic alcoholysis for biodiesel fuel pro more preferably selected from the group consisting of
duction and application of the reaction to oil processing Lipozyme RTL, Lipozyme R RM, NovozymR 435, and mix
(Journal of Molecular Catalysis B: Enzymatic, 2002, 17:133 55 tures thereof.
142) written by Shimada Yuji et al., it is reported that firstly, Said renewable oil comprises biological renewable oils,
the poor solubility of short chain alcohols, such as methanol and preferably comprises Vegetable oil, animal oil and fat,
and ethanol, in the renewable oil raw material is unfavorable waste edible oil and residues of refined oil.
for the reaction; and secondly, the presence of the excess Said vegetable oil comprises castor oil, rapeseed oil, Soy
alcohol may lead to severe deactivation of the enzyme. There 60 bean oil, peanut oil, corn oil, cottonseed oil, rice oil, algae oil
fore, during the process of biodiesel production by the bio and mixtures thereof.
logical enzyme method, batch addition of short chain alcohol Said animal oil and fat comprise fish oil, lard and mixture
is generally adopted to moderate the poisonous effect thereof.
imposed on the enzyme. However, this process is complicated In said short chain alcohol ROH, Risan alkyl group having
in operation and needs long reaction time. Additionally, a 65 1 to 5 carbon atoms, and preferably said short chain alcohol
short chain alcohol is used as an acyl acceptor, and byproduct comprises methanol, ethanol, propanol, butanol and pen
glycerin is produced during the course of reaction, then the tanol.
 US 7,550,278 B2
3 4
Preferably, the reaction molar ratio of said short chain EXAMPLE 4
alcohol to said renewable oil is 3:1 to 5:1, and the amount of
the organic solvent added is 50% to 100% by volume based on Into a triangle flask with a plug, butanol and waste edible
the volume of the renewable oil. oil in a molar ratio of 3:1 (9.65 g waste edible oil) together
Said enzyme reactor may be a triangle flask with a plug, or 5 with 80% by volume of tert-butanol based on the volume of
other suitable enzyme reactors. Preferably, the heating step is the waste edible oil were added and mixed evenly, the flask
carried out in an automatically thermostatic oscillating shaker was placed in an automatically thermostatic oscillating
or in various other bio-reactors suitable for enzyme catalyzed shaker, and the mixture was heated to 40° C., then 10% by
reaction. The preferred temperature for the reaction is 30 to weight of immobilized lipase NovozymR 435 based on the
500 C. 10 weight of the waste edible oil was added to start reaction, and
The advantages of the process of the present invention the reaction was carried out for 7 hours. 9.65 g biodiesel was
comprise the improvement of the lipase reactivity, prolonga produced with an yield of 100%.
tion of the lifetime of the lipase, and remarkable increase in EXAMPLE 5
the yield of biodiesel. This new organic medium reaction 15
system promotes the solubility of the short chain alcohol in
the renewable oil raw material, and effectively decrease the Into a triangle flask with a plug, methanol and Soybean oil
negative influence imposed on the lipase reactivity by the in a molar ratio of 3:1 (9.65 g soybean oil) together with 50%
short chain alcohol. In the organic medium reaction system, by volume of tert-butanol based on the volume of the soybean
the desired short chain alcohol may be added in one step to oil were added and mixed evenly, the flask was placed in an
shorten the reaction time notably, and the yield of biodiesel is automatically thermostatic oscillating shaker, and the mix
increased to 94% or more. Furthermore, the relatively hydro ture was heated to 40°C., then 20% by weight of immobilized
philic organic solvent dissolves part of the byproduct glycerin lipase Lipozyme RTL based on the weight of the soybean oil
and thus the "shield’ effect imposed on the immobilized was added to start reaction, and the reaction was carried out
lipase pores by glycerin is avoided, and the lipase reactivity for 10 hours. 9.1 g biodiesel was produced with an yield of
and lifetime are improved. 25 94%.
The present invention is further illustrated by the following EXAMPLE 6
examples.
EXAMPLE1 Into a triangle flask with a plug, pentanol and rapeseed oil
30 in a molar ratio of 4:1 (9.65 g rapeseed oil) together with
100% by volume of butylbutyrate based on the volume of the
Into a triangle flask with a plug, methanol and rapeseed oil rapeseed oil were added and mixed evenly, the flask was
in a molar ratio of 4:1 (9.65 g rapeseed oil) together with placed in an automatically thermostatic oscillating shaker,
100% by volume of tert-butanol based on the volume of the and the mixture was heated to 50°C., then 30% by weight of
rapeseed oil were added and mixed evenly, the flask was 35 immobilized lipase Lipozyme RTL based on the weight of
placed in an automatically thermostatic oscillating shaker, the rapeseed oil was added to start reaction, and the reaction
and the mixture was heated to 40°C., then 10% by weight of was carried out for 8 hours. 9.17 g biodiesel was produced
a immobilized lipase NovozymR 435 based on the weight of with an yield of 95%.
the rapeseed oil was added to start reaction, and the reaction
was carried out for 6 hours. 9.64 g biodiesel was produced 40 EXAMPLE 7
with an yield of about 100%.
EXAMPLE 2
Into a triangle flask with a plug, ethanol and cottonseed oil
in a molar ratio of 3:1 (9.65 g cottonseed oil) together with
80% by volume of tert-butanol based on the volume of the
Into a triangle flask with a plug, methanol and soybean oil 45 cottonseed oil were added and mixed evenly, the flask was
in a molar ratio of 3:1 (9.65 g soybean oil) together with 20% placed in an automatically thermostatic oscillating shaker,
by volume of tert-butanol based on the volume of the soybean and the mixture was heated to 40°C., then 20% by weight of
oil were added and mixed evenly, the flask was placed in an immobilized lipase Lipozyme(R) RM based on the weight of
automatically thermostatic oscillating shaker, and the mix the cottonseed oil was added to start reaction, and the reaction
ture was heated to 20°C., then 2% by weight of immobilized 50 was carried out for 10 hours. 9.1 g biodiesel was produced
lipase NovozymR 435 based on the weight of the soybean oil with an yield of 94%.
was added to start reaction, and the reaction was carried out
for 24 hours. 9.17 g biodiesel was produced with an yield of EXAMPLE 8
95%.
55 The lipase remained after the reaction of example 1 was
EXAMPLE 3 filtered and used in the next reaction batch, while other reac
tion conditions were kept as same as those in example 1, and
Into a triangle flask with a plug, ethanol and cottonseed oil the lipase was used repeatedly 10 times in the same way.
in a molar ratio of 5:1 (9.65 g cottonseed oil) together with During the 10" reaction batch, 9.62g biodiesel was produced
200% by volume of methyl formate based on the volume of 60 with an yield of 99% after 6 hours of reaction.
the cottonseed oil were added and mixed evenly, the flask was According to the above-mentioned examples, when tert
placed in an automatically thermostatic oscillating shaker, butanol or a short chain fatty acid ester RCOOR' (wherein R
and the mixture was heated to 60°C., then 10% by weight of and Rare independently an alkyl group having 1 to 4 carbon
immobilized lipase NovozymR 435 based on the weight of atoms) is used as an organic medium, and a short chain
the cottonseed oil was added to start reaction, and the reaction 65 alcohol ROH (wherein R is an alkyl group having 1 to 5
was carried out for 12 hours. 9.1 g biodiesel was produced carbon atoms) is used as an acyl acceptor, various renewable
with an yield of 94%. oil raw materials (castor oil, rapeseed oil, cottonseed oil,
 US 7,550,278 B2
5 6
waste edible oil, soybean oil, fish oil, lard, residues of refined 5. The process for producing biodiesel from renewable oil
oil and algae oil, etc.) can be converted into biodiesel effec under lipase catalysis in an organic medium reaction system
tively under a suitable temperature condition upon the addi as claimed in claim 4, characterized in that:
tion of 2 to 30% by weight based on the weight of the renew said vegetable renewable oil comprises castor oil, rapeseed
able oil of an immobilized lipase, such as NovozymR 435 oil, Soybean oil, peanut oil, corn oil, cottonseed oil, rice
(available from Candida antarctica), Lipozyme(R) RM (avail oil, algae oil and mixtures thereof.
able from Rhizomucor miehei) and Lipozyme RTL (available 6. The process for producing biodiesel from renewable oil
from Thermomyces lanuginosus). under lipase catalysis in an organic medium reaction system
What is claimed is: as claimed in claim 4, characterized in that:
1. A process for producing biodiesel from renewable oil 10 said animal renewable oil comprises fish oil, lard and mix
under lipase catalysis in an organic medium reaction system, ture thereof.
wherein a short chain alcohol ROH, wherein R is an alkyl 7. The process for producing biodiesel from renewable oil
group having 1 to 5 carbonatoms, is used as an acyl acceptor, under lipase catalysis in an organic medium reaction system
tert-butanol is used as a reaction medium, and a renewable oil as claimed in claim 1, characterized in that:
raw material is catalyzed by a lipase to synthesize biodiesel 15 said short chain alcohol is selected from the group consist
through a transesterification reaction, characterized in that: ing of methanol, ethanol, propanol, butanol and pen
the short chain alcohol and the renewable oil raw material tanol.
having an alcohol/renewable oil molar ratio of 3:1 to 6:1, 8. The process for producing biodiesel from renewable oil
together with 20-200% by volume of the tert-butanol under lipase catalysis in an organic medium reaction system
based on the volume of the renewable oil, and 2-30% by as claimed in claim 1, characterized in that:
weight of the lipase based on the weight of the renewable the molar ratio of said short chain alcohol to said renewable
oil are added into an enzyme reactor and mixed evenly, oil is 3:1 to 5:1.
the mixture is then heated to 20-60° C. to react for 4-24 9. The process for producing biodiesel from renewable oil
hours to convert the renewable oil raw material into under lipase catalysis in an organic medium reaction system
biodiesel and byproduct glycerin. 25 as claimed in claim 1, characterized in that:
2. The process for producing biodiesel from renewable oil the amount of tert-butanol added as the organic medium is
under lipase catalysis in an organic medium reaction system 50% to 100% by volume based on the volume of the
as claimed in claim 1, characterized in that: renewable oil.
said lipase is a microorganism lipase. 10. The process for producing biodiesel from renewable oil
3. The process for producing biodiesel from renewable oil 30 under lipase catalysis in an organic medium reaction system
under lipase catalysis in an organic medium reaction system as claimed in claim 1, characterized in that:
as claimed in claim 1, characterized in that: the heating step is carried out in an automatically thermo
said renewable oil is a biological renewable oil. static oscillating shaker.
4. The process for producing biodiesel from renewable oil 11. The process for producing biodiesel from renewable oil
under lipase catalysis in an organic medium reaction system 35 under lipase catalysis in an organic medium reaction system
as claimed in claim 3, characterized in that: as claimed in claim 1, characterized in that:
said biological renewable oil comprises Vegetable renew the reaction temperature is in the range of 30° C. to 50° C.
able oil, animal renewable oil, waste edible oil and resi
dues of refined oil. k k k k k
 UNITED STATES PATENT AND TRADEMARK OFFICE
Certificate
Patent No. 7,550,278 B2 Patented: June 23, 2009
On petition requesting issuance of a certificate for correction of inventorship pursuant to 35 U.S.C. 256, it has
been found that the above identified patent, through error and without any deceptive intent, improperly sets forth
the inventorship.
Accordingly, it is hereby certified that the correct inventorship of this patentis: Wei Du Beijin (SN). Dehua
&N. Lillin Beijing (CN); Yuanyuan Xu, Beijing (CN); and Li Wang, Beijing ).
Liu, Beijing
Signed and Sealed this Nineteenth Day of January 2010.
DANIEL M. SULLIVAN
Supervisory Patent Examiner
Art Unit 162F