There is an increasing interest in biodiesel production because of the environmental and economic advantages of this biofuel over traditional fuels derived from non-renewable resources (Akoh et al., 2007). Biodiesel is a renewable, non-toxic and biodegradable fuel (Van Gerpen et al., 2004) defined as the mono alkyl esters of long chain fatty acids derived from vegetable or animal fats, for use in compression-ignition (diesel) engines (American Society for Testing and Material [ASTM], 1989). This fuel can be used pure or in a blend with petroleum-based diesel (Van Gerpen et al., 2004). Currently, large-scale biodiesel production has been done by means of alcoholysis reactions of fats and oils (Reaney et al., 2005). The choice of the suitable alcohol for this process will depend on factors like its cost, availability, toxicity, easy of recovering and recycling, and the amount of alcohol needed for the reaction. Commercially, biodiesel has been produced using methanol as alkyl donor due to advantages like easy recovering and low cost (Van Gerpen et al., 2004). Anhydrous ethanol has also a great potential for biodiesel production in Brazil, one of the largest world ethanol producers (Schuchardt et al., 1998).
Renewable sources from both vegetable (Reaney et al., 2005) and animal (Lee et al., 2002) origin can be used as raw materials for biodiesel production. Microalgal oils have also received much attention as substrate for esters formation (Miao & Wu, 2006). Many agro-industrial processing operations generate a significant amount of lipids as by-products, such as chicken fat, beef tallow and fish oil. This feedstock, usually applied in livestock feeding, is a viable resource to produce biodiesel, and this use is a way for adding value to this material (Feltes et al. 2009). The conversion of used cooking oils and restaurant grease into alkyl esters is another proposal of particular interest in the field of alternative fuels (Lee et al., 2002). The use of biomass as feedstock is an attractive alternative for biodiesel production in tropical countries such as Brazil. The agricultural and farming industries are activities of great importance in many parts of this country. Also, government policies and legislative action may rapidly increase biodiesel production in such region (Ferrari et al., 2005; Meneghetti et al., 2006).
Chemical catalysis is a well established process for biodiesel production (Bournay et al., 2005). The homogeneous alkali-catalyzed transesterification process has been extensively applied to the large-scale synthesis of alkyl esters, especially due to the low cost of base catalysts and their efficiency even at low concentrations. The chemical reaction, however, has some disadvantages as it is energy-intensive, requires several separation/purification steps and generates significant amounts of wastewater to be treated. Additionally, high-temperature alkali catalysis accelerates the oil oxidation, which is a serious drawback when dealing with a feedstock rich in polyunsaturated fatty acids (Damstrup et al., 2006). Also, ester phase processing and waste disposal are great problems when dealing with alkali-catalyzed reactions (Hájek & Skopal, 2010, Van Gerpen et al., 2004).
Many investigations have been conducted for the synthesis of biodiesel mediated by lipases. The enzymatic approach for the modification of oils and fats takes advantage of the specificity of some lipases, leading to high purity products, therefore reducing post processing operations and costs. Due to the mild conditions under which the enzymatic reaction occurs, oil oxidation becomes a negligible problem and less energy is required. Moreover, wastes are minimized and by-products are more easily purified (Gunstone, 1999). It is worth mentioning that the alcohol used in the reaction can be inhibitory to some enzymes (Van Gerpen et al., 2004). Moreover, compared to classical chemical catalysts, the relatively low stability of enzymes in their native state as well as the high cost of commercial lipases are important drawbacks to the use of these biocatalysts in industrial processes (Villeneuve et al., 2000). For these reasons, much effort has been directed for reducing lipases costs therefore allowing the development of competitive enzymatic processes with potential for industrial application. The production of lipases from new sources (Rigo et al., 2010; Wolski et al., 2009), the development of techniques for lipases immobilization (Villeneuve et al., 2000) as well as to perform enzyme-catalyzed reactions in compressed or supercritical fluids, such as propane,
In light of the increasing interest in the development of alternative energy sources, the aim of this section is to make a review of an enzymatic approach for biodiesel production, focusing on a sustainable process. A section of this chapter will be dedicated to the raw materials that have been used as feedstock for biodiesel production, especially in Brazil, outlining the use of agro-industrial residues. We will survey recent researches for the production of lipases with great potential for biodiesel synthesis. The enzyme-catalyzed production of this biofuel in compressed or supercritical fluids will also be reviewed.
2. Biodiesel as fuel
Recently, there are several concerns about the dependence on foreign-controlled fuels sources of finite supply, reserves of petroleum shortages and rising prices of petroleum-based fuels. These problems have encouraged the development of alternative fuels, obtained especially from renewable sources (Reaney et al., 2005) like fats and oils. Oils can be used as liquid fuel for combustion in compression-ignition (diesel) engines directly or in a blend with other fuels. This use, however, has many problems (Rathore & Madras, 2007), that can be overcome by means of pyrolysis, micro-emulsification or transesterification in order to modify raw fats or oils. Transesterification is by far the most used process for oil modification, yielding fatty acid alkyl esters, the so-called biodiesel (Cerveró et al., 2008). This biofuel has a viscosity similar to that of the petrodiesel, as the oil viscosity decreases as transesterification reaction proceeds, as verified for alkyl esters of refined soybean oil (Costa Neto et al., 2004).
Biodiesel possesses several technical and environmental advantages over the conventional diesel. Besides being renewable and of domestic origin, advantages of biodiesel compared to petrodiesel include biodegradability, higher flash point, reduction of most regulated exhaust emissions, miscibility in all ratios with petrodiesel, compatibility with the existing fuel distribution infrastructure, and inherent lubricity (Knothe, 2008; Moser, 2009). Ferrari et al. (2005) produced ethyl esters from neutral soybean oil that were further mixed with petroleum-based diesel and tested as fuel in an energy generator. The diesel oil consumption (0.6981L.h-1) was reduced when this fuel was mixed with up to 20% of biodiesel, when the fuel consumption was 0.6875L.h-1. Technical problems with biodiesel include oxidative stability, cold flow, and increased NOx exhaust emissions (Knothe, 2008).
Solutions to some of the aforementioned problems have included the use of additives, the modification of the fatty acid composition of the feedstock, or mixtures of components with advantageous properties (Knothe, 2008).
In Brazil, the Law 11.097/2005 introduced biodiesel in the energy sources to be used in this country (Agência Nacional do Petróleo, Gás Natural e Biocombustíveis, 2005). Since January 2010, with the National Biodiesel Program, the blend of 5% of biodiesel has been mandatory, according to the Governmental regulation 6/2009 (Conselho Nacional de Política Energética, 2009). The biodiesel produced in Brazil in 2010 was about 2.4 million m3 in 67 facilities whose total capability was 16,414.51m3.day-1 (Agência Nacional do Petróleo, Gás Natural e Biocombustíveis, 2011).
3. Interesterification reactions
Despite of some divergences between authors, acidolysis, alcoholysis (including glycerolysis), and interesterification, all fall under the heading transesterification. Interesterification or ester-ester interchange is the exchange of acyl groups between two esters, namely, two triacylglycerols. Acidolysis is the transfer of an acyl group between an acid and an ester. Alcoholysis is the process of exchanging acyl groups between an ester and an alcohol (Akoh et al., 2007). The most common method now in use for biodiesel production is reversible transesterification reactions in which the oil or fat is reacted with a monohydric alcohol in the presence of a catalyst (Figure 1), where the raw glycerol (glycerin) is obtained as co-product (Knothe & Dunn, 2005).
The glycerin obtained at large amounts and with a low cost in biodiesel production (Yazdani & Gonzalez, 2007) can be used as substrate for the synthesis of monoacylglycerols for application in the food industry, as suggested by Freitas et al. (2009). One has to consider, however, additional steps for products purification should be done when glycerin is obtained from the chemical-catalyzed biodiesel process, in order to assure a high-quality ingredient to human consumption (Hájek & Skopal, 2010).
Alternatively, as shown in Figure 2, methyl acetate can be used as acyl acceptor, where triacetyn, a valuable biodiesel additive, is produced as side product rather than glycerin, which has lower commercial value (Tan et al., 2010). Additionally, alkyl esters can be produced by means of direct esterification of fatty acids with alcohols (Figure 3). In this reaction, water must be removed to increase the yield, as the reverse reaction is the hydrolysis. Direct esterification is possible but not common for biodiesel preparation (Akoh et al., 2007).
Since reactions are reversible, it is advantageous to try to shift the equilibrium toward the synthesis, by using an excess of one of the substrates or continuously removing water eventually formed during the reaction (Villeneuve, 2007).
3.1. Chemical versus lipase-catalyzed reactions
Currently, alcoholysis reaction by means of chemical catalysis is the most used method for the industrial production of biodiesel, especially because of the low cost of the catalysts, and high conversions reached in short time (Van Gerpen et al., 2004; Vicente et al., 1998). Many processes used in the oleochemical industry, however, are energy-intensive, requiring high temperatures (often above 200°C) and pressures. The resulting products generally need to be redistilled to remove impurities formed via thermal degradation. Moreover, highly unsaturated oils are thermally labile (Sheldon, 1996). Additionally, free fatty acids and water from the alcohol or the oil reduce ester yields, as they lead to the formation of soap and further free fatty acids, respectively (Fukuda et al., 2001). In the methanolysis of refined sunflower oil catalyzed by sodium hydroxide, for example, high temperatures (above 60°C) and catalyst concentrations (above 1.5%) led to the production of large amounts of soap (Vicente et al., 1998).
The development of green processes for biodiesel production has received much attention, involving the use of heterogenous catalysts, either chemical (Bournay et al., 2005; Boey et al., 2011) or enzymatic (Adamczak et al., 2009). In this sense, enzymatic processes can provide significant advantages over chemical process for biodiesel production. The mild conditions (pH, temperature and pressure), usually applied in the biocatalyzed reaction, allow energy saving and product quality improvement, because of the minimal thermal degradation of the substrates. Free fatty acids contained in waste oils and fats can be completely converted to alkyl esters, thus making their previous removal unnecessary (Van Gerpen et al., 2004).
In lipase-catalyzed reactions, glycerin can easily be recovered with simple separation processes (Cerveró et al., 2008). Moreover, the purification of fatty acid alkyl esters is simple to accomplish (Fukuda et al., 2001). These characteristics allow the reduction of residues treatment costs and of environmental concerns. Some disadvantages related to lipase-catalyzed reactions for biodiesel production include high cost (Rousseau & Marangoni, 2002), inactivation by acyl acceptors such as methanol depending on the enzyme, inactivation by minor components in the crude oil and waste oils, desorption from immobilization carrier, and fouling in packed bed bioreactors, as discussed by Akoh et al. (2007).
It should be emphasized that enzyme-catalyzed transesterification is a very good option to all chemical-catalyzed reactions; however, it must be developed for its industrial application for esters formation (Kulkarni & Dalai, 2006). In order to use lipases more economically and efficiently in aqueous and non-aqueous solvents, techniques for their modification, to increase their activity, selectivity or stability and to improve their solubility in organic solvents, have been the subject of increased interest (Villeneuve et al., 2007).
4. Lipases as catalysts
Lipases (triacylglycerol lipases, E.C. 220.127.116.11) are carboxylic ester hydrolases with catalytic activity not only in an aqueous solution, but also in nonaqueous solvents (Rousseau and Marangoni 2002). The amount of water present in the reaction mixture influences biocatalysis in several ways. Often optimal water content can be found; further water addition will cause decreased enzyme activity. Water probably activates the enzyme by increasing the internal flexibility of the catalyst molecule. However, water can also act as a substrate in the enzymatic reaction, especially in the reactions of hydrolytic enzymes. This results in side-reactions and lower product yields (Adlercreutz, 1996). The best way to characterize the degree of hydration of a biocatalytic system in organic media is to use the thermodynamic water activity (aw) as the parameter (Adlercreutz, 1996; Halling, 2002).
The optimum water content to the highest enzyme activity depends on the catalyst. The best results for the majority of lipases preparations are with aw values ranging between 0.25 and 0.45, which corresponds to an water content from 0.5% to 1%. The optimal water content, however, can vary up to values as high as 11% (w/v) (Villeneuve, 2007).
4.1. New sources of lipases
Enzymes are produced by microorganisms, plants and animals. Purified enzymes, though expensive, are easy to apply for biotransformation in organic media and their use is increasing. Barriers to the production of industrial enzymes include economic factor, the availability of optimal enzymes and safety issues. The production and purification processes differ for extracellular and intracellular enzymes, liquid and solid culture in the case of fermentation, and enzyme application. Liquid fermentation is useful for the production of both intracellular and extracellular enzymes, especially from bacteria and yeast. It is good for scale-up and reproduction. The use of
Solid state fermentation could be a perfect technology for adding value for low-cost agro-industrial residues aiming the production of enzymes, and for helping reducing pollution problems (Soccol & Vandenberghe, 2003). Salum et al. (2010) produced a lipase from
Griebeler et al. (2009) carried out the solid state fermentation with soybean bran of different microorganisms isolated from agro-industrial sources. They reported the most promising fungi with hydrolytic activity against tributyrin belonged to
Vargas et al. (2008) studied the lipase production by
The use of intracellular lipase as a whole cell biocatalyst immobilized within biomass carrier particles is significantly advantageous since immobilization can be achieved spontaneously during batch cultivation, and in addition, no purification is necessary (Fukuda et al., 2001). For these reasons, Robles-Medina et al. (2009) argued that the use of the whole cell biocatalyst can reduce both the cost of the biocatalyst and dependence on lipase manufacturers. For more details about microbial lipase production, the reader is referred to a recent review on this subject (Treichel et al., 2010).
According to Foglia & Villeneuve (1997), the high cost often associated with an enzymatic process can be overcome by the utilization of plant enzymes, which take the advantage of the lower cost, when compared to microbial lipases, and ready availability. These authors described high activity in synthesis reactions with
5. Substrates and reaction media
The following sections are dedicated to an overview of sources of alcohol, fats and oils that have been used for biodiesel production, focusing on lipase-catalyzed reactions.
5.1. Sources of alcohol
For economic reasons, methanol is most commonly used in transesterification reactions to the commercial production of biodiesel, although other alcohols can also be applied as alkyl donors (Moser, 2009), such as ethanol, propanol, and butanol (Knothe & Dunn, 2005). In general, improved conversion is achieved by adding extra alcohol (Reaney et al., 2005). It is known that largest conversions to methyl esters, for example, are obtained for a methanol to triacylglycerols molar ratio of 6:1 (Vicente et al., 1998). Methanol in excess, however, may lead to lipase inactivation. According to Soumanou & Bornscheuer (2003), particularly with water-miscible alcohol substrates such as methanol, water required to maintain lipase structure can be stripped leading to lower activity and eventual biocatalyst inactivation. This problem can be overcome, for example, by using methyl acetate as acyl acceptor, as already verified in the transformation of soybean oil for biodiesel production catalyzed by the commercial lipase Novozym 435 from
Rodrigues et al. (2008b) presented the alcoholysis of different vegetable oils (soybean, sunflower and rice bran) catalyzed by three immobilized commercial lipases. Novozym 435 from
Higher molecular weight alcohols have lower polarity and therefore their separation from alkyl esters becomes more difficult after reaction completion, which can be overcome by mixing higher alcohols with methanol, glycerol or water (Reaney et al., 2005). Ethanol has a great potential for use in biodiesel production in countries like Brazil, where it is a cheap and abundant commodity produced from the fermentation of sucrose from sugarcane (Rodrigues et al., 2008b). In 2009, Brazil produced 26.10 million m3 of ethanol, whose major producer was by far São Paulo State (58% of the total production) (Agência Nacional do Petróleo, Gás Natural e Biocombustíveis, 2009). In spite of its renewable origin, the ethanolysis reaction has some drawbacks to the industrial production of biodiesel. The yield of fatty acid ethyl esters is less compared to methyl esters and separation of glycerol is the main constrain in the process of ethanolysis (Meher et al., 2006).
Propanol or butanol can also be used in alcoholysis reactions, especially because these two alcohols promote a better miscibility between the alcohol and the oil phases (Iso et al., 2001).
Soumanou & Bornscheuer (2003) were able to produce fatty acid alkyl esters from various vegetable oils (cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) by transesterification with different alcohols (methanol, ethanol, 1-propanol, 1-butanol and isobutanol) using commercial immobilized lipases (AK lipase from
5.2. Sources of fats and oils
Along with vegetable oils, animal fats and oils also have a great potential to be used as substrate for biodiesel production. The choice of the source depends largely on the country and its climate (Knothe & Dunn, 2005), and also on economics (Rodrigues et al., 2008b). A wide variety of feedstock is expected to be used in Brazil for biodiesel production. Currently, the main resource for biodiesel production in this country is soybean oil (
The use of biodiesel has not expanded in developing countries mostly due to the high production cost which is associated with the expensive high-quality virgin oil feedstocks (Hoque et al., 2010). The cost of the raw materials currently represents about 70% of the total production costs (Robles-Medina et al., 2009).
Fats and oils of differing origin have different fatty acid compositions (Table 1). The properties of fats and oils, and hence their commercial value, depend on the fatty acid composition and of the triacylglycerol structure (Sheldon, 1996). For example, a high content of saturated fatty acids in the raw feedstock (i.e. myristic, palmitic, stearic acids) rises turbidity and the cetane number, decreases NOx emissions, and shows better stability. Furthermore, a high ratio of polyunsaturated fatty acids (i.e. linoleic or linolenic acids) has the opposite effect (Cerveró et al., 2008). Knothe & Dunn (2005) discussed in detail the influence of fats and oils compositions on biodiesel properties.
One of the main criteria for the quality of biodiesel is the storage stability which, in turn, depends on the fatty acid composition of the feedstock. Vegetable oil derivatives especially tend to deteriorate owing to hydrolytic and oxidative reactions. Their degree of unsaturation makes them susceptible to thermal and/or oxidative polymerization, which may lead to the formation of insoluble products that cause problems within the fuel system, especially in the injection pump (Meher et al., 2006). Santos et al. (2010) outlined the high amount of unsaturated fatty acids found in fish oil may result in higher oxidation problems. It is also a critical parameter when dealing with phytoplankters and seaweeds rich in long-chain polyunsaturated fatty acids. Autooxidation of unsaturated oils can be limitated by addition of suitable antioxidants (Lands, 2005).a From Liu (2004); b From Gunstone & Harwood (2007).
Fig. 4. Typical fatty acid composition (wt%) found in vegetable and animal oils and fats that may be used for biodiesel production.
Knothe & Dunn (2005) outlined biodiesel is susceptible to start-up and operability problems when subjected to cold temperatures. Biodiesel made from feedstock containing larger amounts of high-melting point long-chain saturated fatty acids tends to have relatively poor cold flow properties. This drawback can be overcome by the transesterification of triacylglycerols with medium chain-length (C3-C8) or branched-chain alkyl alcohols.
Despite of the economic, environmental and social advantages of biodiesel as an energy source, there is concern about the utilization of edible oils as substrates for its production. Taking it into consideration, non-edible and waste oils have been investigated as starting material (Robles-Medina et al., 2009).
5.2.1. Vegetable fats and oils
According to Liu (2004), the range oil content in soybean is 12-30% (dry weight; typical oil content 20%), whose major components are triacylglycerols. There is a large genetic variation in fatty acid composition of soybean oil, mainly resulting from plant breeding. The range of fatty composition (% relative to total oil) among soybean germplasm is palmitic acid, 4-23%; stearic acid, 3-30%; oleic acid, 25-86%; linoleic acid, 25-60%; linolenic acid, 1-15%. This oil contains minor components (phospholipids, unsaponifiable material, free fatty acids, and trace metals) that are reduced after oil refinement. These substances affect lipase activity, as verified in the methanolysis of unrefined soybean oil, containing phospholipids, catalyzed by the immobilized
Palm-base alkyl esters are potential substitutes for diesel fuel, as already investigated by Dors (2011). Palm trees are an attractive alternative feedstock, especially because of their great productivity (610 gallons of oil per acre) (Kurki et al., 2010). These plants produce two types of oils, palm oil from the fibrous mesocarp and lauric oil from the palm kernel. The former contains mainly palmitic (44.0%) and oleic acids (39.2%), while the latter is rich in lauric (48.3%), myristic (15.6%) and oleic acids (15.1%) (Teoh, 2002). The world´s two largest producing nations are Indonesia and Malaysia (United States Department of Agriculture, [USDA], 2011). Batistella et al. (2002) proposed an approach to recover carotenoids, valuable pigments for the food industry, and biodiesel from palm oil by means of suitable techniques.
Castor oil is a unique commercial resource containing ricinoleic acid (12-hydroxy-9-octadecene acid; C18:1, OH) with a major share of up to about 90%. Castor oil is obtained from the seeds of
An alternative feedstock for biodiesel production is the non-edible oil extracted from babassu (
5.2.2. Animal fats and oils
Although animal fat such as beef tallow is considered an industrial residue, and not a renewable source of energy, if compared to oleaginous seeds, there is an increasing interest in its use as feedstock for biodiesel production. Additionally, the use of animal fat from residues as starting material for alkyl esters production plays an important role in the pollution reduction (Krause, 2008). According to Lee et al. (2002), lard is an inexpensive co-product of the meat-packing industry that could be further converted to value-added products such as biodiesel. However, the relatively high contents of saturated fatty acids in lard result in the poor cold temperature properties of its alkyl esters. To improve cold temperature properties, the content of saturated fatty acid in lard could be reduced through a fractionation process before esterification. As a consequence of this procedure, biodiesel from fractionated lard resists forming crystals at low temperatures, thus improving cold-temperature properties.
Chicken fat obtained from processing by-products is a promising alternative feedstock for biodiesel production because of its great availability and low cost (Feltes et al., 2009). Brazil is the third world´s largest producer of chicken meat and the first largest exporter of chicken meat, whose total production was 10.98 million tonnes in 2010 (Associação Brasileira dos Exportadores de Frango, 2011). Boye et al. (2011) studied the methanolysis of low free fatty acids chicken fat by using shells as catalysts. Krause (2008) described the economic feasibility of biodiesel production from animal fats (beef tallow and chicken fat), by using the alkaline methanolic route. The same research group reported the alkali-catalyzed methanolysis of biodiesel from beef tallow in a pilot plant (Cunha et al., 2009). Hoque et al. (2010) demonstrated a high potential of producing economically viable biodiesel from low cost feedstock (beef fat, chicken fat and used cooking oil) with proper optimization of the process parameters.
According to Food and Agriculture Organization of the United Nations [FAO] (2010), capture fisheries and aquaculture supplied the world with about 142 million tonnes of fish in 2008, excluding aquatic plants. Of this, 27.2 million tonnes were used for non-food purposes. The dominant species in marine fishery catches were anchoveta (
Total fish oil production by the five main exporting countries (Peru, Chile, Iceland, Norway and Denmark) in 2009 was 530,000 tonnes, a decline of 100,000 tonnes compared with 2008. Fish-oil prices reached US$950/tonne in March 2010, which was 50 percent higher than an year earlier. For fish oil, almost 85 percent of production was used as an ingredient in fish and shrimp feeds (FAO, 2010). This oil could be applied as substrate for the production of biodiesel (Feltes et al., 2010).
In 2009, the total fisheries production in Brazil was more than 1.24 million tonnes. The Northeast region was the major national producer (34%), but the state of Santa Catarina, at the South, was the largest producer (more than 199,000 tonnes). The sardine (
Santos et al. (2010) investigated the chemical production of biodiesel from oil extracted from the viscera of
5.2.3. Waste fats and oils
Low-cost sources such as restaurant greases (Lee et al., 2002) and soapstock can be used for biodiesel production. These sources, however, are of lower quality than refined vegetable oils, especially because of the high content of free fatty acids, what is a major problem when dealing with transesterification reactions catalyzed by alkali (Knothe & Dunn, 2005). Along with free fatty acids, restaurant grease contains other substances formed during cooking, especially deep-frying (Akoh & Min, 2008), that must be removed prior to alcoholysis in order to a high yield in alkaline-catalyzed reactions (Lee et al., 2002).
The use of vegetable oils as frying oils produces significant amounts of used oils which may present a disposal problem. Their use for biodiesel production has the advantage of their low price. Free fatty acids present in this feedstock can be a drawback when dealing with alkaline catalyzed-processes, but acid or enzymatic reactions can be a practical solution to the production of this biofuel (Knothe & Dunn, 2005). Besides free fatty acids, this starting material contains other degradation products as polymers, as revised by Kulkarni & Dalai (2006). These compounds can affect the catalyst activity, but they can be removed by means of simple filtration. Waste oils have higher amounts of saturated fatty acids, leading to less favourable cold flow properties (Knothe & Dunn, 2005).
Charoenchaitrakool & Thienmethangkoon (2011) investigated the optimum conditions in biodiesel production from waste frying oil using two-step catalyzed process (acid catalysis followed by alkali-catalysis). Although liquid mineral acids can be used to catalyze the modification of an oil with a high free fatty acids content, Silva et al. (2011) pointed out serious drawbacks, such as high corrosiveness, large effluent generation, the impossibility of catalyst reuse, compromise their application. Contrarily, solid acid catalysts appear to be an attractive option; however, the water present or generated during free fatty acids esterification causes the leaching and deactivation of these catalysts.
Coconut and palm kernel distillates containing a high content of free fatty acids (71.3 and 61.2%, w/w as lauric acid), rich in saturated fatty acids, especially lauric acid, a low content of unsaponifiable matter and neutral acylglycerols (Nandi et al., 2005) could be used as starting material for biodiesel production.
5.2.4. Aquatic plants
According to FAO (2010), procedures for the industrial preparation of biofuel not only from fish waste but also from seaweeds are being developed. Aquaculture produced 15.8 million tonnes (live weight equivalent) of aquatic plants in 2008.
Chile was the most important seaweed culturing country outside Asia, producing 21,700 tonnes in 2008, followed by Africa (14,700 tonnes). In 2008, the highest production of cultured seaweed was of Japanese kelp (
The production of algae culture in freshwater was 68,400 tonnes in 2008, especially of
5.3. Reaction media
Several approaches have been made in order to optimize transesterification reactions for biodiesel production in batch systems (Table 2). Many studies are carried out in order to reduce enzymatic processes costs. In an attempt to produce biodiesel from used palm oil and ethanol using immobilized lipases in a solvent-free system, Tongboriboon et al. (2010) verified higher conversions could be achieved using the combination of Lipase AK (from
As can be seen in Table 2, several enzymatic reactions are performed in organic media. An organic solvent is used in order to dissolve the reactants, to enable interesterification rather than simple hydrolysis to occur, because excess water would cause the enzyme to desorb from its support (Cheetham, 1995), and also to improve the mass transfer during the reaction, increasing its rates. In all the solvent-containing biocatalytic systems, however, the nature of the solvent influences the activity and the stability of the enzyme to a large extent. The tendency of solvents to inactivate enzymes does not depend only on their hydrophobicity, expressed as their log
Hydrophilic solvents with log
When using Novozym 435, a commercial immobilized lipase, as catalyst,
* Abbreviations: IM: immobilized. BAUN: Batch Acidolysis Units Novozymes
The key issue in enzymatic methods is to reduce the cost of the catalyst or to improve its operational life (Du et al., 2004). The reuse of the catalyst is crucial to make the process economically feasible to a large scale application (Villeneuve et al., 2000). Furthermore, different reaction media or technologies have been applied for the development of a cost-effective enzymatic system for biodiesel production, as for example the use of low-frequency (10–60 kHz) ultrasound (Rokhina et al., 2009; Kumar et al., 2011), ionic liquids (Gamba et al., 2008). Supercritical fluids have also received much attention, as it will be discussed in the next section.
6. Enzymatic reactions in compressed and supercritical fluids
When a compound is compressed and heat above its critical point, it enters a phase that is referred to as its supercritical phase. The compound in the supercritical region is called as supercritical fluid (Dunford, 2004). An experimental setup used for alkyl esters production with immobilized lipase in pressurized fluids is schematically shown in Figure 4. Potential advantages with supercritical media are the high diffusivity and low surface tension which lead to low mass-transfer resistance (Adlercreutz, 1996).
High temperatures and pressures are needed during the non-catalytic transesterification process (Kulkarni & Dalai, 2006; Silva et al., 2007; Tan et al., 2010). For energy saving, transesterification reactions in supercritical fluids can be enzyme-catalyzed (Adlercreutz, 1996). Moisture content, particle size, and solvent flow rate are important parameters that must be optimized when using supercritical fluid technology for oil and oilseed processing (Dunford, 2004). Supercritical and near-supercritical fluids have been used for enzymatic reactions in a large number of studies. The most widely used medium in this group is supercritical carbon dioxide, whose main advantage is the easy removal after the reaction by just decreasing the pressure. Furthermore, supercritical carbon dioxide has low toxicity compared to organic solvents. As solvent, it resembles hexane and accordingly, hydrophobic compounds can be dissolved, but the solubility of more polar compounds is low, but this can be overcome by addition of organic co-solvents, when necessary. Enzymes are normally not soluble in the supercritical fluids, and therefore heterogeneous systems with solid enzyme preparations are normally used (Adlercreutz, 1996).
Dalla Rosa et al. (2009) indicated the continuous lipase-catalyzed ethanolysis of soybean oil in a packed-bed reactor using compressed propane as solvent as a potential alternative to conventional process for biodiesel production. Good reaction conversions were obtained at mild temperature (70°C) and pressure (60bar), opening possibilities to manipulate process variables as well as reactor configurations towards reaching acceptable yields. Rathore & Madras (2007) investigated the synthesis of biodiesel from edible (palm oil, groundnut oil) and non-edible oils (
A preliminary study comparing conventional and supercritical media for the ethanolysis of palm kernel oil was carried out using two commercial immobilized lipases (Lipozyme IM from
According to Oliveira et al. (2006), the enzyme behaviour in compressed fluids is of primary importance as the loss of enzyme activity may lead to undesirable poor reaction rates and low yields of target products. Enzyme stability and activity may depend on the enzyme species, characteristics of compressed fluid, water content of the enzyme/carrier/reaction mixture and process variables manipulated. These authors investigated the behaviour of two commercial immobilized lipases submitted to compressed carbon dioxide, propane and
Lanza et al. (2004) observed the commercial immobilized lipase from
The commercial exploitation of supercritical fluids technology has been limited to a large-scale biodiesel production. One factor that may contribute to the development of this technology is the study of the phase behaviour of the high-pressure multicomponent systems found in transesterification reactions, as it is essential for the design of any supercritical fluid reaction system. Furthermore, these experimental data are of primary importance for selecting appropriate operational conditions to accomplish enzyme-catalyzed transesterification reactions in supercritical media. The phase behaviour of castor and soybean oils and their fatty acid ethyl esters in carbon dioxide at high pressures was already investigated, for example, by Ndiaye et al. (2006).
Several approaches have been made for a greener biodiesel production by means of biocatalyzed reactions. Lipases production from alternative sources, enzymes immobilization, as well as the use of alternative reaction media, such as supercritical fluids, have appeared as potential alternatives to reach this goal. Besides, the use of alternative feedstock, focusing on agro-industrial residues, seems to be a practical solution to reduce biodiesel production costs while ensuring a minimal environmental impact.