Enzymatic Methods May Improve Vegetable Oil Based Biodiesel Biology Essay

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One of the common types of catalyst is an enzyme catalyst. Enzymes are proteins that increase the rate of chemical reactions, have been used to make foods and beverages. Like all catalysts, enzymes work by lowering the activation energy for a reaction, thus dramatically increasing the rate of the reaction. Enzymes do differ from most other catalysts by being much more specific.

Enzymes have been shown to reduce costs through more efficient processing, the replacement of harsh or toxic chemicals, and the reduction of energy and water use. This realization has dramatically increased the use of enzymes for processing raw materials from plants and animals and has led to new or improved products.

The requirement of removal of catalyst and excessive energy requirements are the major drawbacks for biodiesel being produced chemically using vegetable oil. Enzymatic methods may overcome the problems for the reaction.

Enzyme catalysts have become more attractive recently since it can avoid soap formation, non- polluting and the purification process is simple to accomplish. However, they are less often used commercially because of the longer reaction times, higher cost and denaturation.

Enzymes are generally effective biocatalyst for having substrate specificity in aqueous media.

Biodiesel is produced from lipids, such as vegetable oils and animal fats, by an ester exchange process known as transesterification. This converts the triglycerides of the feedstock into simple fatty acid alkyl esters. Alkaline earth metal hydroxides, especially sodium hydroxide, are the predominant catalysts for the reaction. In cases where the feedstock also contains free fatty acids, an acid-catalyzed step is also required, sulfuric acid being most commonly in use. These inorganic catalysts offer the advantages of affordability and high degrees of substrate conversion.

Enzymes have been proposed to overcome the drawbacks facing the conventional chemically catalyzed biodiesel production methods, and have shown promising result than transesterification using alkali catalysis.

The advantages using enzymes are:

Glycerol can be easily recovered without any complex process,

Free fatty acids contained in the oils can be completely converted to methyl esters and subsequent

Wastewater treatment is not required

Lipase enzyme

Lipases enzyme is potentially attractive catalysts for biodiesel production. Lipases can be used as biocatalyst in the transesterification reaction. A lipase is a high water-soluble enzyme that catalyzes the hydrolysis of ester bonds in water-insoluble, lipid substrates. Lipases thus comprise a subclass of the esterases. Lipases perform essential roles in the digestion, transport and processing of dietary lipids (e.g. triglycerides, fats, oils) in most, if not all, living organisms.

Lipases enzyme is an attractive catalyst for biodiesel production because:

They produce a cleaner product than inorganic catalysts,

It decrease downstream processing costs,

can both esterify free fatty acids and transesterify triglyceride

Easy product separation

minimal wastewater treatment needs

Easy glycerol recovery and the absence of side reactions

However, due to such matters as cost, stability, and rate and extent of reaction, unstable in short chain alcohols lipase catalysis has yet to be applied in commercial biodiesel production.

To avoid the unstable short chain alcohols, the amount of alcohol used must be less than the solubility limit, so that it is not present as a separate phase.

Cost of lipase is the major issue for the industrialization of lipase-mediated bio-diesel production. There are two ways to reduce the lipase cost. One is to reduce the production cost of the lipase, which can be realised through new lipase development, fermentation optimisation, and downstream processing improvement. Another way is to extend the operational life of the lipase, and this can be achieved through enzyme immobilization and alcoholysis reaction optimization.


In order to reduce the cost, immobilised enzymes and whole-cell biocatalysts are introduced to allow enzymes to be held in place throughout the reaction which they are easily separated from the products and be used again which is an efficient process.

In order to use the enzyme catalyst repeatedly, the process of immobilization must be carried out using appropriate method. Immobilization methods are chosen which has the most physical adsorption due to its ease, cheap and nontoxic chemicals, ability to retain the activity, and feasibility of regeneration. Immobilization of the lipase can be achieved by encapsulation in sol-gel or by attachment to a solid surface.

Immobilized lipase increase stability and activity compare with free lipase. Therefore immobilisation is useful. Common immobilization techniques are attachment to solid supports and entrapment within the matrix of a polymer.

If the lipase is immobilized, then it becomes an independent phase within the reaction system, which may easily be retained in the reactor with concomitant advantages in preventing contamination of the products and extending its useful active life.

Increasing the temperature generally increases the rate of lipase-catalyzed reaction per unit amount of active enzyme; however, increasing the temperature also leads to a higher thermal deactivation rate of the lipase itself, thus yielding decreasing amounts of active enzyme. Because immobilization provides a more rigid external backbone for lipase molecule, temperature optima are expected to increase which results in a faster reaction rate.


Type of lipases

Most lipases used as catalysts in organic synthesis are of microbial and fungal origin, such as

Pseudomonas fluorescens (Lipase AK)



Novozym 435

B. cepacia

In addition to the type of lipase, other factors affecting the effectiveness of lipase in the production of biodiesel from triacylglycerol are the water content, temperature, number of cycles (for immobilized lipase) and the type of alcohol and its ratio to oil.

The effect of water content on the production of bio-diesel from soybean oil using lipases from R. oryzae, C. rugosa and P. fluorescens, Novozym 435 and B. cepacia have all shown that enzyme activity was low in the absence of water, which supports the fact that a minimum amount of water is required to activate the enzyme.

With increased addition of water, the amount of water available for oil to form oil-water droplets increases, thus, increasing the available interfacial area. However, since lipases usually catalyze hydrolysis in aqueous media, excess water stimulates the competing hydrolysis reaction.

The amount of water to be maintained in bio-diesel production using immobilized lipase depends on the feedstock, source of lipase, immobilization technique and the type of acyl acceptor. Thus, it was recommended to optimize the water content depending on the reaction system used.

Studies revealed that P.fluorecens lipase shows the highest enzymatic activity.

Acyl acceptors

The alcohol materials that can be used in the transesterification process as an acyl acceptors include methanol, ethanol, propanol, butanol, and amyl alcohol. Among these alcohols, methanol and ethanol are used most frequently. Methanol is especially used because of its lower cost, reactivity and its physical and chemical advantages. Methanol can react with triglycerides quickly and the alkali catalyst is easily dissolved in it. However, methanol is the most toxic and has the most deleterious effect on the biocatalyst activity in comparison to other alcohols. Methanol is easier to produce than ethanol, sustainable methods of methanol production are currently not economically viable and the majority of it is formed from syngas, which is extracted from natural gas (a non-renewable source).

Ethanol can easily be formed from renewable sources by fermentation, which makes the process of biodiesel production, totally 'green'. Therefore, ethanol is more readily accepted for use in a variety of industrial situations.

Producers of biodiesel must decide what types of enzyme and alcohol they will use in their operation. Enzymatic transesterification reaction and immobilization of enzyme experiments can be carried out to determine which lipases are the most useful enzymes and alcohol for biodiesel production.

Since Pseudomonas fluorescens (Lipase AK) shows the highest enzymatic activity, methanol and ethanol experiment were conducted according to a factorial design with respect to four factors including temperature, water content, pH. Changes in all the factors except the substrate molar ratio impacted evidently the production yield of the biodiesel.

Results showed that ethanol had highest conversion of the following factors:

Effect of temperature on the activity of lipase AK.

Effects of pH on the activity of lipase AK.

Effect of water content on the activity of lipases.

Therefore ethanol is best used for acyl acceptors since it gives a higher conversion than methanol.

Vegetable oils

There different types of oil for biodiesel production such as

Sunflower oil




Waste cooking palm


Animal fats have been used for the production of biodiesel. However, due to the high melting points, which are usually near the denaturation temperature of lipase; the reaction has to take place in an organic solvent media to dissolve the solid fat.

Palm oil has the highest yield of around 4000 kg per hectare compared to that of other vegetable oils. Therefore, it would be economically intuitive to consider palm oil as a favourable feed stock for biodiesel production.

Best production of biodiesel for enzyme

Source of enzyme - Pseudomonas fluorescens (Lipase AK)

Amount of enzyme used - 20% wt

Oil - Palm

Acyl acceptor - ethanol

Alcohol to oil molar ratio -18:1

Optimum reaction conditions - 58oc

This gives 98% conversion.