Biodiesel production with various lipases
Liquid fuels have been used for many years as the most dominant and basic fuel for motor engines. However, declining fossil fuel resources as well as the tendency for developing new renewable biofuels have shifted the interest of the society towards finding novel alternative fuel sources. Biodiesel (monoalkyl esters of long-chain fatty acids) has a great potential as an alternative diesel fuel. From an environmental point of view it shows clear advantages over conventional fuel: it comes from renewable sources, and hence does not contribute to new carbon dioxide emission, it is biodegradable, its combustion products have reduced levels of particulates, sulphur oxides, carbon oxides, nitrogen oxides, and therefore, significantly reduces pollution (Al-Zuhair, 2007; Salis et al., 2005). One of the advantages of biodiesel in comparison to other biofuels is that biodiesel can be pumped, stored and handled using the same infrastructure employed for conventional diesel fuel (Robles-Medina et al., 2009). Also, major advantage of biodiesel as an alternative fuel is that its energy content is similar to conventional fuels, so it can be used either on its own or mixed with conventional diesel fuel, with no need of altering existing engines (Bozbas, 2005). European countries have recognized need for alternative fuels and issued the Directive on the Promotion of the use of biofuels and other renewable fuels for transport (2003/30/EC). The Directive stipulates that EU countries should replace 5.75% of fossil fuels with alternative, biofuels until 31. December 2010. This Directive has been amended by Directive 2009/28/EC which also promotes the usage of energy from renewable sources (aims at achieving a 20% share of energy from renewable sources in the EU’s final consumption of energy by 2020). In 2005, the estimated world production of biodiesel was 2.92 million tones of which 87% was obtained in EU. More importantly, between 2000 and 2005 world production increased threefold, indicating that share of biodiesel in global fuel production will significantly increase in future (Mousdale, 2008).
There are several possible processes for biodiesel synthesis: pyrolysis, the use of microemulsions and transesterification. Though pyrolysis, due to reduced viscosity, gives good quality fuel it still produces more biogasoline than biodiesel fuel. Fuel produced by microemulsion creates engine performance problems (Fukuda et al., 2001; Ma and Hanna, 1999). Conventionally, biodiesel is produced by transesterification of triacylglycerol (TAG) and short chain alcohols, commonly methanol, in the presence of an acid or an alkaline catalyst (Marchetti et al., 2007). By this process the flashpoint of biodiesel is lowered and the cetane number is improved (Parawira, 2009). Transesterification is a three-step consecutive reaction, in which diglycerides and monoglycerides are formed as an intermediate compounds (Figure 1. and Figure 2.). Three moles of biodiesel and one mole of glycerol are produced for every mole of TAG that undergoes completely conversion. It is a reversible reaction and accordingly, excess alcohol is used to shift the equilibrium to the products side.
This approach of the biodiesel synthesis has been used on industrial scale for decades, but there are several problems associated with this kind of production. Complex removal of catalyst, excessive energy requirements, recovery of glycerol, undesirable side reactions and the cost of the refined feedstock are the major drawbacks for such chemical process. Not only that the preliminary processing of feedstock is required (to reduce soap formation), but waste water must be treated as well (Fjerbaek et al., 2009). There have been several attempts to overcome these problems and to develop the alternative methodologies for biodiesel production. Nowadays, the biological production of biodiesel with lipases has received great consideration and it is undergoing a rapid development.
The main advantages of employment of lipases as biocatalyst are mild reaction conditions and easy recovery of glycerol without purification or chemical waste production and production of very high purity product. In addition, free fatty acids content in the oil can be completely converted to methyl esters, with no soap formation, and thus increasing the biodiesel yield and reducing the costs for fuel purification. This characteristic of the enzymes allows the usage of materials with high free fatty acids (FFA) or high water content such as non-edible oils, waste cooking oils and industrial waste oil. Transesterification of triglycerides using lipases is considered to be one of the most efficient processes of biodiesel production from waste oil. It is clear that production process for alkaline method is more complex than enzymatic procedure as it is shown in Figure 3. and Figure 4.
Despite numerous advantages, enzymatic processes have drawbacks such as: low reaction rate, enzyme cost for industrial scale use in comparison to alkali catalyst, low enzyme stability in the presence of excess methanol (Bajaj et al., 2010; Fjerbaek et al., 2009). In order to make enzymatic transesterification competitive on industrial scale there are several issues that have to be addressed: solvent engineering, lipases immobilization, selection of acyl acceptor, and selection of the reactor system. In this chapter we will try to review offered solutions to the current problems and to look for new perspectives in the industrial application of enzymatic biodiesel synthesis.
2. Lipases as biocatalysts in biodiesel synthesis
Lipases (triacylglycerol hydrolase, EC 184.108.40.206.) are enzymes that catalyze the hydrolysis of carboxylic ester link in the triacylglycerol molecule to form free fatty acids, di- and monoglycerides and glycerol. Although their natural function is to catalyze hydrolysis of ester links, they can also catalyze the esterification, the creation of this link between alcohol hydroxyl groups and carboxyl groups of carboxylic acids. Therefore, since they can catalyze hydrolysis, alcoholysis, esterification and transesterification they have a wide spectrum of biotechnological applications.
Lipases are also highly specific as chemo-, regio- and enantioselective catalysts. Thanks to direct evolution and protein engineering it is possible to enhance catalytic potential of lipases and to ‘tailor’ them to specific application and process conditions, enabling further expansion of their industrial use (B van Beilen and Li, 2002; Kirk et al., 2002). Among lipases of plant, animal and microbial origins, most commonly used are microbial lipases. They have numerous advantages over lipases from animals and plants. Using microorganisms it is possible to achieve a higher yield of enzymes, and to genetically manipulate the producing strain in obtaining a low-cost lipase with desired properties for the conversion of natural fats and oils into biodiesel. In addition, the enzymatic yield is independent of potential seasonal variations and it is possible to achieve rapid growth of microorganism in low-cost culture media (Gupta et al., 2004).
|Waste cooking palm oil||Methanol||4h||79.1%||Halim et al. (2009)|
|Soybean oil||Ethanol||10h||70%-100%||Rodrigues et al. (2010)|
|Methanol||24h||97%||Royon et al. (2007)|
|Soybean oil||Methanol||solvent-free||12h||68%-95%||Guan et al. (2010)|
|Soybean oil||Methyl acetate||solvent-free||14h||92%||Du et al. (2004)|
|Sunflower oil||Methanol||solvent-free||24h||90%-97%||Dizge et al. (2009)|
|Waste cooking oil|
|Sunflower oil||Methyl acetate||solvent-free||12h||"/>95%||Ognjanovic et al. (2009)|
|Methanol, Propanol,||solvent-free||7h||91.5%||Kose et al. (2002)|
|Butanol, Amyl alcohol|
|Rapeseed oil||Methanol||solvent-free||24h||91.1%||Watanabe et al. (2007)|
|Rapeseed oil||Methanol||12h||95%||Li et al. (2006)|
|Jatropha seed oil||Ethyl acetate||solvent-free||12h||"/>90%||Modi et al. (2007)|
|Sunflower oil||Methanol||n-hexane||24h||"/>80%||Soumanou and Bornscheuer (2003)|
|Sunflower oil||Methanol||solvent -free||24h||"/>90%|
|Waste cooking oil||Methanol||10h||91.08%||Chen et al. (2009)|
|Waste oil||Methanol||24h||92,80%||Li et al. (2009)|
|Waste cooking oil||Methanol||solvent-free||72h||"/>90%||Ying and Chen (2007)|
|Jatropha seed oil||Methanol||tert-butanol||48h||94%||Kumari et al. (2009)|
|Rice bran oil||Methanol||solvent-free||120h||80,20%||Kamini and Iefuji (2001)|
|Soybean oil||Methanol||solvent-free||90h||"/>80%||Kaieda et al. (2001)|
Reaction system for lipase is a two-phase system consisting of aqueous phase with dissolved enzyme and an organic phase with dissolved substrate. Lipases have a specific reaction mechanism due to the fact that active site of the enzyme is covered by amphiphilic peptide loop that acts like a lid. This disables the substrate molecule to bind to the enzyme active site, resulting with negligible activity of lipases in aqueous solutions (in the absence of an interphase or organic solvent). When contact occurs with a lipid/water interface, lid undergoes a conformational rearrangement which renders the active site accessible to the substrate (Schmid and Verger, 1998).
A large number of lipases from different sources have been utilized for biodiesel synthesis as shown in Table 1.
2.1. Lipases immobilization
One of the main obstacles for industrial application of lipases is the high cost of biocatalysts. Therefore, immobilization of lipases, which allows their reusability, is a necessity to make them more attractive for industrial biodiesel processes. The aim of immobilization is to enhance lipases properties such as thermostability and activity in non-aqueous media, and to improve handling, recovery and recycling of biocatalyst. Recycling of immobilized enzymes greatly reduces the cost of the production, so the most promising immobilization supports and methods could make the enzymatic biodiesel production competitive to chemical processes.
By definition, the immobilization of enzymes is localization or confinement of an enzyme on to a solid support or on a carrier matrix. There are a number of supports that can by used for immobilization and the selection depends on several factors: thermal stability, chemical durability, mechanical strength, lipase type, type of the reaction system, ease of regeneration, loading capacity and cost (Jegannathan and Abang, 2008). Generally, immobilization is accompanied by changes in enzyme activity, optimum pH, temperature and stability. The productivity of an immobilized system is evaluated through both, activity and stability of a biocatalys. Often, the loss of enzyme activity is compensated by enhanced operational stability, which in overall makes the immobilization method a viable procedure. Methods for enzyme immobilization can be classified as physical adsorption, entrapment, covalent bonding and encapsulation, each with its advantages and disadvantages. Different immobilization techniques of lipases used as catalysts in biodiesel production are summarized in Table 2.
Adsorption is the most widely used method for lipase immobilization primarily because procedure is easy, conditions are mild and the method itself is cheap (Hilal et al., 2006; Nie et al., 2006; Yagiz et al., 2007). This technique involves no toxic chemicals, supports can be regenerated for repeated use, and there is no major activity loss. Various supports have been used such as: textile membrane, alumina, ceramics, sepharose, sepadex, cellulose, hydrotalcite, different type of zeolites, celite, silica gel, polyethylene, polypropylene and others. The nature of support strongly dictates catalytic properties of the lipase such as activity, selectivity, stability (Knežević et al. 2004). Yang et al. explored the influence of pore diameter of nonpolar and weakly polar resin on degree of immobilization and reported that the degree of immobilization increases with increasing pore diameter. Also, the higher lipase activity was obtained employing nonpolar resins (Yang et al. 2006). It has been documented that lipase activity is generally higher with hydrophobic support. At low ionic strength, hydrophobic areas surrounding the active site are adsorbed on hydrophobic support, leaving stabilized the open form of the lipase (Mateo et al. 2007; Petkar et al. 2006). Shah and Gupta immobilized
|Adsorption||Celit||Jatorpha oil||Ethanol||98%||Shah and Gupta (2007)|
|Adsorpiton||Cotton membrane||Waste oil||Methanol||92%||Nie et al.|
|Adsorption||Hydrotalcite and zeolites||Waste oil||Methanol||92,80%||Yagiz et al. (2007)|
|Adsorption||Nonpolar resin||Soybean oil||Methanol||97,30%||Yang et al. (2006)|
|Adsorption||Toyonite 200-M||Safflower oil||Methanol||Iso et al.|
|Adsorption||Polypropylene support, Accurel MP 100||Mahua oil||Ethanol||96%||Kumari et al. (2007)|
|Cross-linked enzyme aggregates (CLEAs)||None||Mahua oil||Ethanol||92%||Kumari et al. (2007)|
|Protein-coated microcrystals (PCMCs)||None||Mahua oil||Ethanol||99%||Kumari et al. (2007)|
|Soybean oil||Methanol, ethanol||65%||Noureddini|
et al. (2005)
|Entrapment||Phyllosilicate sol-gel matrix||Restaurant grease||Methanol, ethanol||87-95%||Hsu et al. (2002)|
|Adsorption||Macropouros polypropylene||Soybean oil||Methanol||98%||Salis et al. (2008)|
|Entrapment||Phyllosilicate sol-gel matrix||Restaurant grease||Ethanol||96%||Hsu et al. (2004)|
|Encapsulation||Silica aerogels||Sunflower oil||Methyl acetate||Orcaire et al. (2006)|
|Covalent bond||Aldehyde-Lewatit||Soybean oil||Ethanol||75%-100%||Rodrigues|
et al. (2010)
|Covalent bond||Silica -PVA composite||Babassu oil||Ethanol||98%||Freitas et al. (2009)|
|Covalent bond||Novel microporous polymeric matrix (basically copolimer of styrene cross-linking with divinilbenzene)||Sunflower oil||Methanol||63,80%|
|Soybean oil||55,50%||Dizge et al. (2009)|
|Waste cooking oil||50,90%|
|Covalent bond||Chitin activated with hexamethylenediamine (HEMDA)||Butyric acid||Butanol||187||Gomes et al. (2004)|
|Covelent bond||Amino-functionalized magnetic nanoparticles||Soybean oil||Methanol||94%||Xie and Ma (2009)|
|Covelent bond||Silica-PVA composite||Palm oil||Ethanol||98%||Moreira et al. (2007)|
Lipases immobilized by entrapment are more stable than physically adsorbed lipase. Entrapment of lipase includes capture of the lipase within a matrix of a polymer. The porose nature of the matrix allows the retention of the enzyme within the support, also enabling substrate and product diffusion. This method uses a relatively simple procedure and a number of natural and organic supports: alginate, agarose, gelatin, phyllosilicate sol-gel matrix. A variety of methods have been documented. For example, Meunier and Legger developed a procedure for entrapment of lipase from
Encapsulation is the confinement of enzyme within a porous membrane forming a bilayer (Jegannathan et al. 2008). In a way, it provides a cage, which prevents enzyme leaching making the resulting immobilized enzyme a highly reusable biocatalyst. Encapsulation can be carried out by using natural polymers, such as alginate and carrageenan, synthetic polymers (photo-cross linkable resins and polyurethane polymers), acrylic polymers, hydrogels, microemulsion based gels and those obtained by sol–gel methods (Yadav and Jadhav, 2005). For the use in biodiesel synthesis lipases from
The main advantage of covalent immobilization is the irreversible bonding of the lipase to the support matrix, thereby preventing leaching of the enzyme in the reaction system. The immobilization by covalent bonding is the most complex one. Since the supports don’t have the reactive groups but hydroxyl, amino, amide and carboxy groups, they have to be activated for immobilization of enzymes. Therefore, covalent immobilization usually consists of two stages: activation or modification of the support and attachment of the enzyme on the activated support. Activation of the support implies chemical reaction between support and an activating agent, creating new (usually electrophilic) groups on its surface that show a great affinity towards functional groups (usually nucleophilic) of the enzyme. According to the presence of certain groups, appropriate method of chemical activation is chosen. It is very important that the immobilization of lipase by covalent attachment to a support should involve only functional groups of the lipase that are not essential for its catalytic activity (Knežević et al. 2006; Prlainović et al. 2011). When it comes to supports with carboxy groups, enzymes are immobilized on the polymers through their amino groups, forming a peptide bond, with the presence of carbodiimide as a carboxy activator group. Covalent immobilization via the carboxy group of supports is often used in immobilization of lipase on the polymer derivates of acrylic acid. It must be noted that these active derivatives enable mild covalent coupling of lipase. Polymers containing amino groups can be activated by the introduction of the diazo groups. Enzymes are then easily linked through their α- or ε-amino groups and less by sulfhydryl, hydroxyl, imidazole or carboxy groups. One of the most commonly used methods of activating supports with amino groups is with glutaraldehyde. Recently different authors have focused their attention on developing a large number of supports containing epoxy groups. These groups can react with various nucleophilic groups of amino acids residues (amino, hydroxyl or sulfhydryl group) at very mild conditions: neutral pH and temperatures between 4 and 25°C (Mateo et al. 2000; Bezbradica et al. 2009). In this way, a vast number of very strong bonds are formed, and a considerable increase of stability is achieved due to multiple bonding. In addition, polymers containing epoxy groups can be easily modified by other activating agents such as glutaraldehyde, if the lipase activity decreases due to the formation of undesirable interactions between the enzyme and supports due to short distance (Bayramoglu et al. 2005). Immobilization methods are highly specific and should be optimized for a particular lipase support system. For the use in biodiesel synthesis, lipase from
2.2. Pretreatment of lipase
Pretreatments of lipase by various methods have a goal of improving enzyme activity, enzyme stability and, especially important for biodiesel synthesis, to improve methanol tolerance. Researches have shown that when immobilized enzyme is successfully pretreated enzyme activity is increased and the ability of the enzyme to resist deactivation by methanol is higher, thereby enabling multiple reuses of enzymes for industrial production. Generally, pre-treatment reagents can be classified as: 1) substrates or their analogues; 2) organic solvents; 3) salts; 4) enzyme lyoprotectants such as crown ethers (Lu et al. 2010). Treatments with different reagents have, more or less, the same goal, keeping the conformation of lipases in its active form, by making the conformational change of active site from closed to open form. The most commonly used method is the usage of substrates or their analogues as pre-treatment reagents. They have the role of enhancing enzyme activity in organic solvents by molecular imprinting. The formed complex enzyme-imprinter keeps the enzyme in its active conformation and therefore retains higher activity in the nonaqueous environment as compared to the nonimprinted enzyme (Rich et al. 2002). Lu et al. pretreated immobilized lipase form
3. Key operational variables
A number of different factors influence the enzymatic biodiesel synthesis: oil source, reaction temperature, choice of acyl acceptors, acyl acceptors to oil molar ratio, amount of water in the system or water activity, and presence of organic solvent in the mixture. Optimal parameters for enzymatic transesterification vary depending on the origin and type of lipase, type of oil source, and reactor type.
Reaction temperature may vary from 23 to 50°C. Optimal temperature for methanolysis of sunflower oil is 50°C when
One of the key factors of enzymatic ester synthesis is the water content in the system. Lipases need an optimal small amount of water to maintain the activity in the organic media. Nevertheless, increased water concentration has an unfavourable effect on the equilibrium conversion, since it promotes reverse reaction of hydrolysis. The amount of water in the system should be a compromise between minimizing hydrolysis and maximizing lipase activity for the transesterification reaction and it should be determined for a particular reaction system (Chowdary, 2002; Noureddini et al., 2005). Many studies have shown that immobilized enzymes show highest activity in low water system. Tamalampudi et al. showed that, in biodiesel synthesis using lipase from
There are many possible raw materials with a potential to obtain biodiesel. Generally, the main feedstock for biodiesel production can be divided in: 1) Vegetable oils such as sunflower oil (Dizge et al., 2009; Modi et al., 2007), soybean oil (Guan et al., 2010; Rodrigues et al., 2010), rapeseed oil (Li at al., 2006; Watanabe et al., 2007), jatropha oil (Shah and Gupta, 2007; Tamalampudi et al. 2008), cotton seed oil (Royon et al.,2007); 2) Animal fats such as tallow, lard (Da Cunha et al.,, 2009; Ngo et al. 2008); 3) Waste cooking oils and industrial waste oils (Chen et al., 2009; Halim et al., 2009).The fatty acid composition of animal fats is not favourable for biodiesel production, since they contain predominantly saturated fatty acids. The limited supplies of animal fats and high melting points of obtained esters, which require addition of very powerful freeze protectants, make animal fats substrate of minor importance. On the other hand, vegetable oils are renewable and virtually inexhaustible energy source. Depending on availability of raw materials in different countries, various oils have been used: soybean oil is predominantly used in the United States, rapeseed oil and sunflower oil are the main feedstock for biodiesel synthesis in many European countries. Coconut oil and palm oil are used in Asian countries (Demirbas, 2009). Figure 5. and Figure 6. shows the usage of oils for biodiesel production as well as world production by feedstock in 2007 (International Grains Council, 2007).
However, edible oils are not in surplus supply and the cost of oil sources accounts for a large part in biodiesel production. In order to make the biodiesel production viable the solution is to develop a production based on waste cooking oils where no competition with food production takes place. But the amount of waste oils alone is not sufficient to meet demands. The optimal solution is to use non-edible oils which can not be used for human consumption because of the presence of some antinutritional factors, or toxic components. The most suitable oils are those from crops with the highest productivity pre hectare, low production cost and that can grow on waste land, such as
Biodiesel produced from oils that have high content of unsaturated fatty acids show higher pour and cloud point properties, making it appropriate for both cold and warm weather. However, the quality of such biodiesel is lower since it has lower cetane number and combustion temperature (Demirbas, 2008; Ghaly, 2010). Then again, biodiesel produced from oils with long chain fatty acids (longer than 18 carbons) has high cetane number and combustion temperature, but low cloud and pour point and greater viscosity. So the choice of feedstock should be a compromise between the unsaturation and the length of fatty acid chain (Robles-Medina et al., 2009).
3.1. Solvent engineering and acyl acceptor selection
The majority of enzymatic syntheses of biodiesel are performed in organic solvents. The yield of biodiesel is greatly influenced by the type of organic solvent present in the reaction system. Immobilized lipases showed high degree of efficiency in the presence of non-polar solvents (Soumanou and Bornscheuer, 2003). The polar, less hydrophobic solvents are not suitable for biocatalytic processes since they can distort water microlayer around the enzyme influencing its native structure, thereby, leading to denaturation. It has been shown that the highest biodiesel yield with lipase from
However, despite the promising results, use of solvents should be avoided because of their toxicity and flammability, damaging effects on the environment and consequential requirement for solvent removal. From the economic point of view, the use of organic solvents is unfavorable due to the necessity of their removal from the final product which further increases production cost. Thus, to enable enzymatic processes to be competitive, enzymatic solvent-free systems have been developed. It has been shown that there are many benefits in using the solvent-free system in comparison with the organic solvent system, including the costs reduction and the improvement in the process control (Kose et al. 2002; Selmi and Thomas, 1998). The main drawback in industrial implementation of lipases into the solvent-free biodiesel synthesis is rather low enzyme stability in the presence of excess methanol, since several studies reported that a high methanol concentration could lead to serious inactivation of lipase (Kose et al. 2002; Royon et al. 2007). Methanol is the most popular alcohol used in transesterification process because of its relatively low price in comparison to other alcohols. In a typical methanolysis reaction, the reaction mixture consists of two phases due to low solubility of methanol in oil, leading to the inactivation of enzyme and the decreased fatty acid methyl ester yield. To minimize the enzyme inactivation, the solvent-free reaction systems with a stepwise addition of methanol have been developed. The most common way is a three step addition of methanol in accordance with the reaction dynamics, enabling conversions as high as 98.4% after 48 h (Shimada et al., 1999). Several other studies confirmed that the three-step addition of methanol is superior to the one-step addition (Shimada et al., 2002; Watanabe et al., 2001, 2002). A group of researches has performed the methanolysis of plant oils by lipase immobilized from
Several studies have focused their attention on branched and long chain alcohols. Experiment showed that increase of the number of carbon atoms increased the cetane number as well as heat content of the fuel. Also, fatty acid esters of secondary or branched-chain alcohols can be used as fuel additives since they decrease the solidification point, and consequently, the high cloud point and pour point (Salis et al. 2005; Watanabe et al. 2007). Kose et al. dealt with the alcoholysis of cotton seed oil with primary and secondary alcohols by using Novozyme 435 lipase. They analyzed the effect of alcohol types on alcoholysis on cotton seed oil indicating that the highest yield was obtained with isoamyl alcohol (Kose et al., 2002). Ognjanović et al. investigated the influence of methanol, 2-propanol and
4. Reactor configuration and industrial application
Important aspect of lipase implementation in biodiesel industrial production is development of an efficient immobilized reactor system. On a laboratory scale most commonly used systems are stirred batch reactors. The advantage of this reactor system is easy handling and control, as well as the simple configuration of the reactor. There are numerous studies on enzymatic transesterification in various reactor configurations with the emphasis on determining the best operational parameters, but there isn’t sufficient information on scale-up systems (Du et al., 2007; Talukder et al., 2009). Packed bed reactor (PBR) systems are frequently applied for the large-scale enzymatic reactions. They enable continuous production with high efficiency, and reduced shear denaturation of the biocatalyst. PBR are more suitable for long lasting and industrial production since it allows the reuse of enzyme without the need of a prior separation and the immobilized enzyme is subjected to fewer shear stress, in comparison to stirred batch reactors,. The main advantage of packed bed reactors over fluidized bed reactors or continuous stirred tank reactor is the higher conversion per weight of catalyst. Basic construction of packed bed reactors (on a laboratory scale) is a glass column, packed with immobilized lipases with the reaction mixture placed in thermostat water bath, and pumped upward through the column by a peristaltic pump. Attention has turned to determining the operational and hydrodynamic conditions that maximize the yield in an industrially feasible immobilized system. Chen et al. reported an efficient system for continual production of biodiesel fuel by methanolysis of waste cooking oil (WCO) using immobilized
Packed bed reactor has been tested in laboratory scale by Severac et al. for biodiesel production from high-oleic sunflower oil using
Similar PBR was optimized by Hailm et al. for continuous synthesis of biodiesel from waste cooking oil with methanol using
It was previously reported that, due to internal mass transport limitation, packed bed reactor don’t seem to be an option for solvent free enzymatic FAME production (Fjerbaek et al., 2009). There are several problems to address: due to the high viscosity of solvent free systems pressure drop becomes significant. In order to minimize the pressure drop packed bed reactors need to operate at low flow velocities, the size of carrier must be increased or solvent must be added. With increasing carrier particle diameter the pressure drop decreases, but the internal mass transfer rate in biocatalyst decreases, which can affect the overall reaction rate. Despite these apparent limitations several PBR system have been developed for solvent free reactions (Hajar et al., 2009; Hama et al., 2007; Shimada et al., 2002; Ognjanović et al., 2009). The usage of solvents only increases production costs as they have to be removed and purified for recycling. Shimada et al. developed methanolysis of vegetable oil in a PBR packed with
A very interesting and important study was conducted by Sotoft et al. They carried out the process simulation and economical evaluation of an enzymatic biodiesel production plant (Sotoft et al., 2010). Since there is, to date, only one plant in the world that uses enzymatic transesterification of biodiesel these kinds of studies are very important to make a correct evaluation of the industrial potential of a particular process. The study evaluated several important and relevant scenarios for enzyme catalyzed biodiesel production processes. Enzymatic biodiesel production from rapeseed oil and methanol has been investigated for solvent-free and cosolvent production processes. Study included two different production scales (8 and 200 mil.kg biodiesel/year) and different enzyme prices: current price 762.7€/kg enzyme and assumed price of enzyme in the future 7.627€/kg enzyme. The process simulations were carried out in Aspen Plus and Aspen Icarus Process Evaluator. Based on the simulations, the solvent free process is viable for a larger scale production of 200 mil.kg biodiesel/year with the current enzyme price. Also the continuous operation is the only realistic option with stepwise addition of methanol. Byproduct sale is also an important part of process economy. For a solvent free operation the cost of raw materials is distributed as: 50% enzyme, 47% oil and 3% methanol. The influence of enzyme cost is lower for cosolvent operation due to the improved enzyme performance. The estimated product price was 0.73-1.49€/kg biodiesel with the current enzyme price and 0.05-0.75€/kg with the enzyme price for a future for solvent-free processes.
The production and consumption of biodiesel will inevitably rise in future due to low environmental impact, ease of handling, and possibility of use without need for major adjustments of existing engines of motor vehicles. Although the majority of biodiesel manufacturers still employ a base-catalyzed process, biocatalytic methods based on activity of various microbial lipases offer several advantages, such as mild reaction conditions and high selectivity of biocatalysts in comparison with chemical catalysts. These advantages have good prospects to cause significant increase of economical feasibility of biodiesel production, since mild conditions decrease energy costs, while high selectivity leads to avoiding of unwanted by-products formation, simpler downstream processing, and easier separation of valuable by-product – glycerol. The main obstacle of biocatalytic process application on industrial level is high costs for enzyme formulations, but simultaneous efforts in optimization of different parameters of biocatalytic process led to significant increase of productivity. Various microbial lipase producers with strong affinity towards catalysis of methanolysis reaction and low susceptibility towards presence of methanol have been identified during previous two decades. The stability of lipases has been further increased by pre-treatment of lipases, selection of adequate immobilization procedures or use of whole-cell biocatalysts, leading to prolonged activity of biocatalyst. Detrimental effects of methanol on lipase activity opened new area of investigation – selection of alternative acyl-acceptors, such as higher or branched alcohols, and esters. Investigations in this area led not only to prolonged stability of biocatalyst, but to additional valuable by-products and even biodiesel of improved fuel properties. Additionally, the reduction of oil costs has been performed by development of processes with waste oil and further increase of productivity was achieved by application of novel reactor designs. It can be concluded that promising novel findings obtained in optimization of lipase-based processes of biodiesel production indicate that biocatalysis has great potential in biodiesel synthesis.
The authors are grateful to the Ministry of Science, Republic of Serbia for financial support (Project III 46010).