This document reviews biodiesel production methods using chemical and biological catalysts. Biodiesel can be produced via transesterification, where triglycerides from oils react with alcohol to form esters and glycerol. This reaction is catalyzed by acids, bases, or enzymes. Key process variables that affect conversion rates include the type of catalyst, substrate, temperature, solvent, molar ratios, and glycerol byproduct removal. While base catalysis is most common, acid and enzyme methods allow processing of low-quality feedstocks. Alternative acyl acceptors like methyl acetate and dimethyl carbonate also show promise. Overall, optimizing catalysts, substrates, and process conditions can improve biodiesel

1. Biodiesel production using chemical and
biological methods – A review
of process, catalyst, acyl acceptor, source and
process variables
B. Bharathiraja, M. Chakravarthy, R. Ranjith Kumar, D. Yuvaraj, J. Jayamuthunagai,
R. Praveen Kumar, S. Palani
Presented By: Bijaya K. Uprety
PhD (Biotechnology) student

2. Introduction
Biodiesel has gained good reputation in the
catalogue of renewable energy
Produce reduced toxic emission and can be
blended with diesel and used in conventional
engines too
Its direct use into vehicle  impossible due
to higher viscosity, low volatility & reactivity
of unsaturated hydrocarbon chains present
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3. In 1990 Scientist found ways to reduce viscosity and molecular weight of
vegetable oil
Most notable one were 1. Pyrolysis 2. Tranesterification
Pyrolysis  expensive and yield of undesirable products.
Hence, research was extensively made on the transesterification and resulting fuel
was called as biodiesel.
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4. Chemically, it is known as Fatty Acid Alky Esters (FAAE) and the alkyl group is
decided by the acyl acceptor used for the reaction
Compared to vegetable oil 1/3rd reduction in MW, 1/10th reduction in viscosity
and contains 10- 11% oxygen (w/w) which enhances its combustion process
The versatility of the biodiesel  various methods by which it can be produced
commercially
It can be done by varying any one of the following: (i) Oil/Fat source (ii) Catalyst
(iii) Acyl acceptor and (iv) Solvent
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5. Biodiesel Production
Produced by transesterification (also called
as alcoholysis) is the reaction of fat or oil
with an alcohol to form esters and
glycerol.
Usually a catalyst is used to mediate the
reaction and bring out quicker reaction
rate 1. Acid catalyst 2.Base catalyst 3.
Enzyme catalyst
After transesterification of triglycerides,
the products are a mixture of esters,
glycerol, alcohol, catalyst and tri-, di- and
mono-glycerides.
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6. Typical Production of Biodiesel from veg. oil
1.http://www.lct.ugent.be/sites/default/files/events/Lecture%202%20Studies%20on%20esterification%20of%20Free%20Fatty%20
Acids%20in%20biodiesel%20production.pdf.
If feedstock contain <4% FFA:
Trans-esterification reaction:
Oil + Alcohol  Ester + Glycerol
Catalyst: NaOH, KOH, &
carbonates, H2SO4, HCl, lipases etc.
(Acid catalyzed rxn is slow).
If feedstock contains >4% FFA:
i. Before trans-esterification, FFAs
are converted into soaps and
removed from the Oil
(triglycerides).
ii. Application of the Acid Catalysis
method to trans-esterify the
triglycerides & esterify the FFAs in
parallel in the same reactor.
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7. Acid catalyzed process
The transesterification process is catalyzed by acids and these catalysts give very
high yields of alkyl esters, but the reactions are very slow
The homogeneous acid catalysts are H2SO4, HCl, BF3, H3PO4 and some organic
sulfonic acids  upto 99% conversion has been reported
When excess of acid is added, better conversion of triglyceride is obtained
Advantage: Direct biodiesel production from low cost lipid feed stocks, such as
waste cooking oil, greases etc. These oil sources have FFAs level of 46%
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8. Generally, liquid based acid catalyst are used
Acid addition  protonation of the carbonyl group of the ester  results in
carbocation
Produces the tetrahedral intermediate (after a nucleophilic attack of the alcohol ) 
which eliminates glycerol to form the new ester and regenerates the catalyst H+
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Reaction Mechanism

9. 8
Protonation
Carbocation
Tetrahedral
intermediate
Removal of
glycerol &
regeneration
of proton

10. Advantage of Solid heterogeneous acid catalysts
Insensitive to FFA content of the oil so regular removal of biodiesel and
by-product from the reactors not required Enabling easy recovery and reuse
of solid catalyst which in turn reduces corrosion problems
Disadvantage
 The reactions are slow
 Typically requires temperatures above 100 °C
 Requires more than 3 h to reach complete conversion
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Solid heterogeneous acid catalysts have the potential to replace liquid acid
catalysts

11. Alkali catalyzed process
Most widely used homogeneous base catalysts are NaOH, CH3ONa and KOH
Presence Moisture enhance soap formation  consumes the catalyst and
reduces the efficiency & increase viscosity
Reactions with alkali catalysts are found to perform quick than the acid
catalyzed reactions
Standard value of the reaction to take place is 60 0C, but depending on the oil
source and catalyst, different degrees of conversion is obtained at various
temperature ranging from 25 to 120 0C
Heterogeneous solid alkali catalysts are basic zeolites, alkaline earth metal
oxides and hydrotalcites
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12. The first step is the reaction of base with the alcohol, producing an alkoxide and
the protonated catalyst
The nucleophilic attack of the alkoxide at the carbonyl group of the triglyceride
generates a tetrahedral intermediate, from which the alkyl ester and the
corresponding anion of the diglyceride are formed
The latter deprotonates the catalyst, thus regenerating the active species, which
is now able to react with a second molecule of the alcohol, starting another
catalytic cycle.
Diglycerides and monoglycerides are converted by the same mechanism to a
mixture of alkyl esters and glycerol
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13. 12
Alcohol Base Alkoxide Protonated catalyst
Nucleophilic attack of alkoxide
at carbonyl group Tetrahedral intermediate
Alkyl ester
Anion of diglyceride
Deprotonation
Reacts with another
alcohol for new cycle

14. Super critical methanol process
A fluid is considered supercritical when its temperature and pressure go above
its critical point
They can effuse through solids like gas and dissolve materials like liquid
Problems with transesterification process
• Time consuming process
• Separation of catalyst and Saponified impurities
• Reduced catalyst efficiency & its high consumption
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15. SCM have hydrophobic nature with a lower dielectric constant form a single
phase oil/methanol mixture
Since the reaction is catalyst free, purification of biodiesel is easy, environment
friendly and completes in 2–4 min
These problems are eliminated in the non-catalytic supercritical methanol
method of transesterification
Saka and Kusdiana preheated methanol (350 0C, 24 h) enough to convert
rape seed oil to methyl esters
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16. Hence it is not a viable option for industry level commercialization
The uses of co-solvents such as carbon dioxide, hexane, propane, etc. are being
investigated to reduce the operational parameters and make the process
economical
Yield of methyl esters increases with increase in molar ratio of oil to methanol
Due to severe reaction conditions and high operational costs, this method
suffers few disadvantages
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17. Enzyme catalyzed transesterification – lipases
Lipase dependent catalysis have certain advantages over conventional catalysis
Use of lipase easy removal of glycerol by-product and environmentally
friendly
Problem with alkali based catalyst- Difficult glycerol recovery & treatment of
highly alkaline waste water
Extracted Lipase and some Lipase producing Microorganisms (mostly Fungi) are
immobilized in biomass support particles and used as catalytic beds to obtain
prolong use
Commercial names of lipases are
Novozym, Lipolase, Lipozyme, Lipomax,
Lumafast 16

18. 17

19. A. Extracellular lipase
Extracellular enzyme catalysis Requires Downstream processing
technique (enzyme extraction) & Immobilization to ensure repeated
use
Preparation of lipase solution
Commercial Lipase (0.5 g) + 5.0 ml of water (stirred for 1 h)
followed by centrifugation at 3500g for 10 min
Supernatant was used as enzyme solution after dilution
with water
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20. Immobilization of lipase enzyme and optimization
Many techniques and different carriers have been employed for
immobilization of lipases to produce biodiesel
Carrier used: Both hydrophilic and hydrophobic
Commonly used: Kaolite particles, macroporous resin,
functionalized nanoscale SiO2 spheres
Most lipases exist in two confirmations an open (dominative in
hydrophobic interface) and a closed confirmations (dominative in water)
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21. The covalent attachment of lipase on styrene-divinyl benzene-
polyglutaraldehyde support has been found to be twice stable than lipase
immobilized on to styrene-divinylbenzene beds by hydrophobic
interactions
In closed conformation Flap exposes hydrophilic side  towards water
and hydrophobic side towards catalytic side
In presence of triglyceride the hydrophobic part of lipase (catalytic
site) changes in confirmation, opens and adsorbs the substrate in open
configuration
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22. B. Intracellular lipase or whole cell biocatalyst
for biodiesel fuel production
Use of extracellular lipase expensive due to downstream process
Some of the species of lipase producing microorganisms having the
ability to act as whole cell biocatalyst have been studied
Rhizopus oryzae, Mucor meihei , Candida antartica, Candida rugosa and
Candida cylindracea  Studied for their use as whole cell biocatalyst
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23. B1. Whole cell immobilization or bed preparation
Immobilized R. oryzae cells were the first whole cell biocatalysts used in
the process of transesterification
Cells were cultivated under normal conditions and then immobilized
within biomass support particles (BSP)
In some cases immobilized culturing of cells has been carried out
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24. Hama grew R. oryzae in 100 ml basal
media (30 0C, 24 hr)
For immobilized cell culturing
Transferred to air lift bioreactor
(30 0C) 101 Basal medium
with olive oil (30 g/l) and 24,000
Biomass support particle (BSP)
Aeriation  cause liquid & particle
mixing
After cultivation, the
BSP immobilized cells
were separated from
the culture and
stabilized (packed bed
reactor or bottle flask)
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25. Use of novel acyl acceptors in lipase catalyzed
process
Apart from the regular acyl acceptors like methanol and ethanol,
researchers have also proposed the use of other novel acyl
acceptors such as methyl acetate, ethyl acetate and dimethyl
carbonate
Use of excess lower chain alcohols deactivation of the immobilized
lipase
Use of less amount of methanol or use of Alternative solvents
such as hexane and t-butanol  Can solve the problem
Use of Alt.
solvents is
expensive
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26. Use of Methyl acetate
Du et al. Replaced alcohol with methyl acetate High yield (92%,
molar ratio of oil: methyl acetate: 1:12) was obtained
The yield was obtained for crude and refined soybean oil
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27. Use of Ethyl acetate
Lipase B from C. antartica immobilized on acrylic resin reacted
with crude oil of Jatropha, Karanj and Sunflower oil in presence of
ethyl acetate
Yield of 91.3%, 90% and 92.7% resp. obtained
Molar ratio; EA/oil : 11/1
Temp: 50 0C, 12 hr
By-product obtained was triacetin, a valuable molecule which has
wide spread application
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28. Use of dimethyl carbonate
For use of methyl acetate and ethyl acetate large
amount of compound is required (1:12 of oil/methyl acetate;
1:11 of oil/ethyl acetate)
DMC is a neutral, odorless, cheap, non corrosive, non-toxic
compound that exhibits good solvent properties
With DMC reaction remains in positive product formation
side (Biodiesel) as the product is CO2 which escapes as gas.
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29. Process variables
Basic parameters that
are considered to affect
the conversion rates
Type of
lipase
Type of
substrate
Temperature
& pH
Water,
solvent &
glycerol
content
Molar
ratio of
substrates
Purity of
reactant
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30. Selection of lipase/organism
Selection of the lipase mainly depends on
 Whether the system is a solvent involved system/solvent independent
system
Type of fatty acid involved
 The lipase is to be used intra-cellular or extra-cellular along with other
reaction parameters
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31. An organic solvent such as n-hexane, s-butanol, petroleum ether are added to a
system Increase the miscibility between the triglyceride and methanol 
Increasing the catalytic efficiency of lipase
Reactions can also be carried out in a
solvent free system
Lipases from various microbes give various results in different systems
Triacyl glycerol (TAG) and free fatty acids (FFA) present in the oil decides the
activity of the lipase
Specificity of lipases for biodiesel synthesis
refers to their region specificity or
specificity with respect to the length of
hydrocarbon chain of fatty acid
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32. Whole cell biocatalysts are considered better  cheaper
Disadvantage of reduced
conversion rates compared to
immobilized lipases
The lipases selected for the catalysis are those that display wide substrate
specificity. Examples are lipases from pseudomonas and candida species
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33. Selection of Substrates for biodiesel production
Selection of Lipids source
Selection of solvents
Selection of acyl
acceptors
 Proper selection of source important
 High amount of phospholipid in the oil
gives low flame: lipase pretreatment,
oil degumming/ dewaxing required
 Refined oil gives higher yields but it is
costly
 Methanol, ethanol, isopropanol
 Iso-butanol, 2-butanol and 1-butanol
 methyl acetate, ethyl acetate and di
methyl carbonate
Ideal solvent should ensure the good
solubility of oil and alcohol & also
maintain the enzyme stability (so
mixture of solvent better)
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34. Glycerol effect
Glycerol formation Influence the transesterification process by inactivating
the enzyme
Inactivation of enzymes could be due to
1. Formation of hydrophilic coating of glycerol  due to binding of glycerol
to supporting matrix where lipase is bound reduce the access of enzyme
to TAG
2. Decrease in the water activity of the enzyme
Glycerol has to be continuously removed
 Add hydrophilic compounds such as silica gel to the system
 Washing immobilized lipase after transesterification with isopropyl alcohol
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35. Conclusion
Though many plants and microbes are known to produce oil, most of
them are not able to produce oil sustainably
Production of biodiesel involves different methods
Use of biological catalyst has recently got interest but still at naïve
stage
Various process variables should also be considered
many researches are ongoing to optimize each of the production steps
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