7
Biochemical catalytic production of biodiesel
Abstract
Biodiesel, a renewable fuel, has great potential in fulfilling an ever-increasing transport fuel demand. The conventional alkaline process for biodiesel production is energy-consuming and generates undesirable by-products. Lipases, due to their catalytic efficiency and specificity, have emerged as a great tool for converting a wide range of feedstock oils to biodiesel. Through enzymatic catalysis, applying lipases (in both free and immobilized form), is viable to producing a cleaner and greener biodiesel under milder conditions, since it produces fewer wastes than the conventional chemical process. Limitations associated with the industrial viability of enzymatic methodology are mainly due to high enzyme production and purification costs, the relatively slower reaction rate, and lipases inactivation caused by methanol and glycerol, which may be overcome by molecular technologies.
This chapter summarizes several recent developments that display the current state and perspectives for enzymatic biodiesel production. Also, it indicates the key operational variables that influence lipase activity and stability together with the technological solutions for industrial implementation of the enzymatic-biodiesel production process.
Keywords
Biodiesel; FAEE; FAME; Glycerol; Lipases; Transesterification7.1. Introduction
Biofuels are not only able to fully replace the global demand for fuels, but they can cover an increasing part of the same, which greatly reduces the fossil fuel dependence, prolonging the life of the existing oil reserves, thus making softer the transition to a predictable world scenario without fossil fuels. In this respect, Fig. 7.1 (Calero et al., 2015), shows the main, currently existing methods to transform triglycerides into applicable diesel-engine biofuels. Among them, vegetable oil transesterification currently is the most common method for the biodiesel production. This relatively simple process is capable of reducing oil viscosity at a level (4–5 mm2/s) similar to conventional fossil diesel. Because of its simplicity, this process has been extensively studied. Moreover, it is the only industrial method currently applied to convert vegetable oils into biodiesel (Juan et al., 2011).
Biodiesel has emerged as an environmentally friendly and renewable alternative fuel to petroleum-based fuels. Reserves of conventional petroleum-based fuels, which are only located in certain parts of the world, are rapidly diminishing. Because of ever-increasing prices of fossil diesel and environmental concern due to emission of toxic compounds on its combustion, many countries across the world are encouraging the use of biodiesel as a transport fuel.
Biodiesel is defined as a mixture of long-chain Fatty Acids Methyl Ester (FAME) derived from renewable lipid sources, such as vegetable oil or animal fat, which can be used in compression ignition engines with little or no modifications (Demirbas, 2009). The most usual method to transform oil into biodiesel is transesterification (Fig. 7.2), which can be carried out using different catalytic systems (Marchetti et al., 2007; Demirbas, 2008; Calero et al., 2015) or in supercritical conditions (Demirbas, 2007).
Biodiesel can be produced locally using various feedstocks, depending upon its availability in a particular region, and thus provides energy security. The high oxygen content in biodiesel allows its complete combustion in engines, so that exhaust emissions have lower amounts of particulates, hydrocarbons, and gases such as CO, CO2, and SOx, making this fuel environmentally friendly (Atabani et al., 2012; Robles-Medina et al., 2009; Rounce et al., 2010). Biodiesel, due to its high flash-point of around 150°C, is very safe for transport and storage (Yusuf et al., 2011). It is a viable option as an alternative to petroleum-based fuels because it can be used in its pure form or blended with petroleum-based fuels, without modification of existing engines or with only minor modifications (Singh et al., 2014). In addition, biodiesel possesses better lubricant properties than fossil diesel, which enhances engine yield and extends engine life (Vasudevan and Briggs, 2008).
The catalysts currently studied for biodiesel production may be grouped in some of the following categories: homogeneous or heterogeneous catalysts (that may be carried out through alkaline or acid catalysis); or enzymatic methods. In the last decade, particular attention has been dedicated to the use of lipases as biocatalysts for biodiesel production (Gog et al., 2012; Zhao et al., 2015). The pros and cons of using lipases as biocatalysts compared to alkaline and acidic catalysts for biodiesel production are summarized in Table 7.1 (Gog et al., 2012). In general, lipases perform their catalytic activity in more gentle conditions when a higher variety of triglyceride substrates, including raw materials, waste oils, and fats with high levels of free fatty acids (FFA), are used. Furthermore, biodiesel separation and purification is much easier, resulting in a more environmentally friendly process.
This process became a research hot-spot in academic communities during last 10 years. An increasing number of scientific publications, including articles, review papers, book chapters, patents, and conference abstracts have been published (Zhao et al., 2015) as shown in Fig. 7.3.
7.2. Lipases
Lipases are found in animals, plants, and microorganisms, and they play a key role in the metabolism of oils and fats. Lipases take part in the deposition, transfer, and metabolism of lipids (Villeneuve et al., 2000). Lipases are hydrolases (EC 3.1.1.3) that act on carboxyl ester bonds in triglycerides to yield fatty acids and glycerol. Lipase catalyzes this reaction at the lipid–water interface. The structure of lipase has a central L-sheet with an active site consisting of a serine on a nucleophilic elbow placed in a groove of the structure. This groove is covered by a peptide lid; when lipase comes in contact with a lipid–water interface, this lid undergoes conformational changes, making the active site accessible for the acyl moiety (Jegannathan et al., 2008; Villeneuve et al., 2000). Lipases have both hydrolytic as well as synthetic activity and, thus, can take part in various industrially important reactions like esterification and transesterification (alcoholysis and acidolysis). Lipases from fungi and bacteria are easy to produce in bulk amounts because of their extracellular nature (Gupta et al., 2004).
Table 7.1
Comparison of enzymatic technology versus chemical (alkaline and acid) technology for biodiesel production
| Parameter | Enzymatic process | Chemical process | |
| Alkaline process | Acid process | ||
| FFA content in the raw material | FFA are converted to biodiesel | Soaps formation | FFA are converted to biodiesel |
| Water content in the raw material | It is not deleterious for lipase | Soaps formation. Oil hydrolysis resulting more soaps | Catalyst deactivation |
| Biodiesel yield | High, usually around 90% | High, usually >96% | High yields (>90%) only for high alcohol to oil molar ratio, high catalyst concentration, and high temperature |
| Reaction rate | Low | High | Slower than for alkaline process |
| Glycerol recovery | Easy, high-grade glycerol | Complex, low-grade glycerol | Complex, low-grade glycerol |
| Catalyst recovery and reuse | Easy, reusability proved but not sufficiently studied | Difficult, neutralized by an acid partially lost in postprocessing steps | Difficult, the catalyst ends up in the by-products No reusable catalyst |
| Energy costs | Low, temperature: 20–50°C | Medium, temperature: 60–80°C | High, Temperature: >100°C |
| Catalyst cost | High | Low | Low high cost of equipment due to acid corrosion |
| Environmental impact | Low, wastewater treatment not needed | High, wastewater treatment needed | High, wastewater treatment needed |
Lipases are widely used in the processing of fats and oils, detergents and degreasing formulations, food processing, the synthesis of fine chemicals and pharmaceuticals, paper manufacture, and the production of cosmetics and pharmaceuticals (Hasan et al., 2006).
Figure 7.3 Publications in the years of 2000–2013 found in the Web of Science database by the keywords “biodiesel and lipase” separated by document types.
Lipases (EC 3.1.1.3) are powerful tools that in addition to hydrolysis reactions, also catalyze various synthetic reactions including esterification, transesterification, and aminolysis. Lipases have excellent catalytic activity and stability in nonaqueous media and their specificity, regioselectivity, and enantioselectivity can be successfully used for many applications in organic synthesis, including kinetic resolution and asymmetric synthesis (Gog et al., 2012).
Lipases can be divided into three classes based on their specificity and/or selectivity: regio- or positional specific lipases, fatty acid–type specific lipases, and specific lipases for a certain class of acylglycerols (mono-, di-, or triglycerides). In terms of regioselectivity, lipases have been divided into three types: sn-1,3-specific (hydrolyze ester bonds in positions R1 or R3), sn-2-specific (hydrolyze ester bond in position R2), and nonspecific (do not distinguish between positions of ester bonds to be cleaved). Most known lipases are 1,3-regiospecific with activity on terminal positions.
Another important aspect was the acyl migration phenomenon inside the triacylglycerol molecule reported by several studies (Du et al., 2005). Substrate specificity of lipases is determined by their ability in distinguishing different structural features of acyl chains such as the nature of the acyl source (eg, free acid, alkyl ester, glycerol ester), length, position of double bonds, configuration of double bonds, and the presence of branched groups. Thus, lipase selection is one of the most important/influential factors for biodiesel production from various renewable raw materials.
Lipases produced commercially are mostly of microbial origin. Submerged culture and solid-state fermentation are widely used methods for commercial lipase production. Lipase- producing microorganisms such as bacteria, fungi, and yeasts are isolated and screened for their lipolytic activity (Gupta et al., 2004; Li and Zong, 2010). Based on the lipolytic activity, the microorganism with high activity is selected for commercial lipase production. Lipase production depends upon a number of factors such as carbon and nitrogen sources, pH, temperature, dissolved oxygen, agitation, and metal ions (Sharma et al., 2001). Lipase production can also be induced by providing lipids as a carbon source.
Purification strategy includes concentration of culture medium by ultrafiltration or ammonium sulfate precipitation followed by further purification using sophisticated techniques such as affinity chromatography, ion-exchange chromatography, and gel filtration (Gupta et al., 2004). Several novel techniques such as membrane processes, immunopurification, hydrophobic interaction chromatography, and column chromatography are applied for purification of lipases (Saxena et al., 2003). The production and purification schemes of lipases, for large-scale application, should be high-yielding, rapid, and inexpensive (Gog et al., 2012; Dossat et al., 2002).
7.3. Enzymatic production of biodiesel
7.3.1. Extracellular and intracellular lipases
There are two major categories of enzymatic biocatalyst: extracellular lipases and intracellular lipases. In the case of extracellular lipases, the enzyme has previously been recovered from the live-producing microorganism broth and then purified, while the intracellular lipase remains either inside the cell or in the cell walls. The major producing microorganisms for extracellular lipases are Mucor miehei, Rhizopus oryzae, Candida Antarctica, and Pseudomonas cepacia (Gog et al., 2012).
Previous studies reported the use of free lipases for biodiesel production have principally focused on the screening of lipases (Shah and Gupta, 2007) and on the investigation of the factors that influence the reaction rate (Szczesna-Antczak et al., 2009). Soluble lipases have the advantages of an easy preparation procedure and its low cost. However, they can be used only once in many cases, as they are inactivated after the first use. The improvement of immobilization technologies have provided lipases with an enhanced level of reusability and operational stability, resulting in higher conversion rates and shorter reaction times, respectively (Ranganathan et al., 2008).
The major disadvantage of producing biodiesel by means of extracellular enzymes is the relatively high cost of the lipase due to complex separation and purification procedures. By contrast, using microbial cells that are producing intracellular lipase as whole-cell biocatalysts, acceptable ester yields can be achieved at a lower cost. However, we must take into account that using intracellular lipases means that the process is slower than when using extracellular lipases. Until now, mainly the intracellular lipases investigated were biocatalytic systems based on Rhizopus oryzae yeast.
7.3.2. Lipase immobilization
In recent years, the production of biodiesel using immobilized lipases (IL) has attracted great interest. Significant progresses have been made on both of the immobilization techniques and process development for IL-mediated biodiesel production. ILs show many advantages over soluble, or free lipases (FLs), for the large-scale application in biodiesel production (Li et al., 2012), such as easy recovery and reuse, higher adaptability for continuous operation, less effluent problems, greater pH and thermal stability, and higher tolerance to reactants and products. However, the current ILs still show several drawbacks for industrial applications, including: (1) loss of enzymatic activity during immobilization, (2) high cost of the carriers, (3) low stability in oil–water systems, and (4) the requirement of novel reactors for well mixing and maximizing oil-to-biodiesel conversion.
Many materials have been explored in literature to immobilize lipases, including various polymer resins, celite, silica, ceramics (Zhao et al., 2015), carbon nanotubes (Tan et al., 2012), magnetic particles (Ren et al., 2011), and microspheres (Zhang et al., 2012b). However, for industrial applications, the carrier material must be of low cost. In addition, the immobilization procedure should be easy to perform with a high active-lipase recovery rate, and the IL activity must be maintained for a long running-time. Generally, these goals can be achieved by: (1) improving the immobilization technologies, (2) optimizing the transesterification process, (3) developing novel bioreactors, and (4) intensifying the process integration to reduce the operation cost.
Different immobilization methods can be applied for lipases used in biodiesel production: adsorption, cross-linkage, entrapment, encapsulation, and covalent bonding (Jegannathan et al., 2008). Several examples (Zhao et al., 2015) are shown in Fig. 7.4. Depending on the type of interactions between enzymes and carriers, these techniques can be further classified into irreversible and reversible immobilization techniques (Zhang et al., 2012a). Covalent bonding, entrapment, and cross-linking are the most commonly used procedures for irreversible immobilization of lipases. Physical adsorption and various noncovalent bondings, such as affinity bonding and chelation bonding, are well-known reversible immobilization procedures. Each immobilization technique has its own merits and inevitably some disadvantages for lipase immobilization.
7.3.2.1. Immobilization of lipase by physical adsorption
Adsorption is a commonly used method to immobilize lipase. Several noncovalent interactions are involved in this immobilization, including nonspecific physical adsorption, biospecific adsorption, affinity adsorption, electrostatic interaction (also ionic binding), and hydrophobic interaction (Zhao et al., 2015). Compared with other immobilization techniques, adsorption immobilization is advantageous in the following aspects (Zhang et al., 2012a): (1) mild conditions and easy operation, (2) relatively low cost of carrier materials and immobilization procedure, (3) no requirement of chemical additives during adsorption, (4) easy regeneration of carriers for recycling, and (5) high lipase-activity recovery.
7.3.2.2. Immobilization of lipase by ionic bonding versus covalent bonding
In the immobilization process by ionic bonding, the enzymes are bound through salt linkages. The carriers typically contain ion-exchange residues such as polysaccharides and synthetic polymers (Zhang et al., 2012a).
The ionic bonding process can be easily performed, but the interactions between lipase and carrier are much stronger than physical adsorption. Compared with the covalent bonding method, ionic bonding can be conducted under a much milder condition; therefore, the ionic binding method causes little changes in the conformation and the active site of the lipase, retaining lipase activity in most cases. However, the binding forces between enzymes and carriers are less strong than that of covalent binding, and leakage of enzyme from the carrier may occur in substrate solutions of high ionic strength or upon variation of pH (Zhao et al., 2015).
7.3.2.3. Immobilization of lipase by entrapment or encapsulation
Entrapment immobilization refers to the capture of enzymes within a polymeric network or microcapsules of polymers that allows the substrate and products to pass through but retains the enzyme. After entrapment, lipase proteins are not attached to the polymeric matrix or capsule, but their diffusion is constrained. Compared with physically adsorbed lipases, entrapment-mobilized lipases are more stable. Entrapment immobilization is relatively more simple to perform than covalent bonding, while the activity of lipases is maintained. However, when entrapped lipases are used for biodiesel production, the conversion rate is relatively low. In addition, the entrapped lipases also show relatively low stability (Zhang et al., 2012a).
7.3.2.4. Immobilization of lipase by cross-linking
Immobilization of lipase by cross-linking refers to the process of immobilizing the enzyme via the formation of intermolecular cross-linkages. It can be achieved by the addition of bi- or multifunctional cross-linking reagents such as glutaraldehyde. This immobilization technique is usually support-free and involves joining enzymes to each other to form a three-dimensional structure (Murty et al., 2002). Lipase can be directly immobilized from fermentation broth and recovered as cross-linked enzyme aggregates (CLEAs). The formed CLEAs demonstrate significantly high stability in aqueous solutions within a broad range of pH and temperature values (Lai et al., 2012).
In spite of all the advantages, cross-linking reactions are usually performed under relatively harsh conditions, such as using cross-linking reagents that can change the conformation of lipases and potentially lead to significant losses of activity. Other disadvantages associated with cross-linking immobilization are low immobilization yields and the absence of desirable mechanical properties. To address these two concerns, cross-linking is always coupled with other immobilization techniques such as adsorption.
7.3.2.5. Commercialization of immobilized lipase for biodiesel production
Until now, thoroughly investigated commercial immobilized lipases are Novozym 435 (Hernandez-Martin and Otero, 2008), Lipozyme TL IM (Wang et al., 2008), and Lipozyme RM IM (Aguieiras et al., 2013). All of them are extracellular enzymes. The most widely used are Novozym 435, from Candida antarctica, immobilized on a macroporous acrylic resin; Lipozyme RM IM, from Rhizomucor miehei, immobilized on an anionic resin; and Lipozyme TL IM, from Thermomyces lanuginosus, immobilized on a gel of granulated silica.
7.3.3. Variables affecting the enzymatic transesterification reaction
Crucial factors affecting productivity of enzymatic biodiesel synthesis are shown in Fig. 7.5 (Szczesna-Antczak et al., 2009). To achieve the economic viability, the suitable raw materials and lipase have to be chosen. The latter can be modified to improve stability and catalytic efficiency. These steps are followed by selection of organic solvent, optimization of substrate molar ratio, temperature, water activity, pH of enzyme's microenvironment, and the highest-permissible glycerol concentration in reaction products (the so-named subparameters).
7.3.3.1. Lipid source
Lipases are competitive catalysts in comparison with acids and alkali because a wide variety of triglyceride substrates can be used for the enzymatic synthesis of biodiesel. An economically viable solution for biodiesel production is to use waste or useless fats as a triglyceride source (Gog et al., 2012).
Currently, the main raw materials used to produce biodiesel are the vegetable oils extracted from oleaginous plants. The cost of these materials currently represents about 70% of the total production costs. This means that the most suitable vegetable oils are those from crops with the highest productivity per hectare or low-cost oils such as waste oils. These days, the high fossil fuel prices, the collapse of food for biodiesel initiatives, and concerns about increased levels of CO2 emissions in the atmosphere have all created awareness of the need for alternative fuel solutions. Microalgae have optimistically emerged as one of the potential lowest-cost feedstocks for biodiesel production (Robles-Medina et al., 2009).
Fats and oils may be characterized according to their physical (eg, density, viscosity, melting point, refractive index, etc.) or chemical properties (eg, acidity, iodine index, peroxide index, saponification index, etc.). These parameters will influence the biodiesel quality. This means that the fatty acid profile of the oil influences the quality of the biodiesel produced (Demirbas, 2008).
7.3.3.2. Acyl acceptor
Various types of acyl acceptors, alcohols-primary, secondary, straight and branched-chain, esters can be employed in transesterification using lipases as catalysts.
Alcohols are the most frequently used acyl acceptors, particularly methanol and, to a lesser extent, ethanol. Other alcohols can be used, eg, propanol, butanol, isopropanol, tert-butanol, branched alcohols, and octanol, but the cost is much higher. Regarding the choice between methanol and ethanol, the former is cheaper, more reactive, and the fatty acid methyl esters (FAME) are more volatile than those of the fatty acid ethyl esters (FAEE). However, ethanol is less toxic and it can be considered more renewable because it can be easily produced from renewable sources by fermentation of agricultural feedstocks. In contrast, methanol is mainly produced from nonrenewable fossil sources, such as natural gas. Methanol also inhibits lipases. Regarding their characteristics as fuels, FAME and FAEE show very slight differences (Demirbas, 2008).
A stepwise addition of methanol was the most common strategy to avoid lipase inactivation (Chen et al., 2009). Using a different acyl acceptor as methyl acetate or ethyl acetate, the lipase inactivation is also avoided (Jeong and Park, 2010). Another strategy for solving the problem of lipase inactivation by methanol is the use of organic solvents (Iso et al., 2001), but difficulties in solvent recovery make these methods less competitive at an industrial scale.
7.3.3.3. Temperature
Enzymatic transesterification is generally performed at a lower temperature than the chemical reaction to prevent loss of lipase activity. Lipases from different sources show varying optimum temperature in the range of 20–70°C for their activity. Moderate temperature requirements by lipase-catalyzed transesterification make this process less energy-intensive. An increase in temperature increases the enzyme activity up to optimum temperature, beyond which denaturation of enzyme occurs, thereby decreasing its activity. With the increase in reaction temperature, initial reaction rate also increases, thus reducing the time taken for conversion. However, because of the enzyme denaturation beyond optimum temperature, conversion efficiency decreases.
The deciding factor for optimum temperature of the lipase-catalyzed reaction includes immobilization, stability of lipase, alcohol to oil molar ratio and the type of solvent. In the continuous process, temperature is the key operational factor (Fjerbaek et al., 2009). In conclusion, the optimum temperature for the enzymatic transesterification process results from the interaction between the operational stability of the lipase and the rate of transesterification reaction (Gog et al., 2012).
7.3.3.4. Water content
Water is essential to maintain lipase conformation and it also increases the interfacial area between aqueous and organic phase where lipases act (Tan et al., 2010b). Water content in reaction mixture can be determined by either water activity (aw) or as weight percentage of feedstock oil. Water activity (aw) is the ratio of vapor pressure of a given system to that of pure water (Szczesna-Antczak et al., 2009). Excess water takes part in transesterification reactions and leads to hydrolysis, and thus can reduce the yield of alkylesters. Optimum water content for the transesterification reaction is therefore very important. The optimum water content in the reaction depends upon the lipase and feedstock used, immobilization technique employed, and type of solvent (Lu et al., 2009).
Water content sensitivity is crucial for transesterification, because optimum water content should have striking balance, so that it should minimize the hydrolytic reaction and maximize the activity of lipase. Different water substitutes like tert-butanol and surfactants when added in reaction, could not match the yield from the water-added reaction. Water took part in subsequent hydrolysis and esterification. Water also diluted the ethanol, which had an inhibitory effect on the lipase (Lu et al., 2009). Lipases from different sources showed different responses toward water content.
7.3.3.5. Inhibition by alcohol
Alcohol is a popular acyl acceptor for the transesterification reaction for biodiesel production. Methanol is the most widely used alcohol for transesterification because of its low chain length, which results in a high biodiesel yield, and also it is being least expensive among the alcohols.
Thus, the ratio of methanol to oil is a critical parameter in optimization studies. Various lipases have shown a different level of tolerance toward methanol. Most of the researchers have optimized the methanol to oil molar ratio in the range of 3:1 to 4:1 for lipase-catalyzed conversion. Some lipases have shown optimum activity at higher methanol to oil ratio. To overcome methanol inhibition, various alternatives have been suggested by researchers, including stepwise addition of methanol, the use of other acyl acceptors, use of solvent, and use of methanol-tolerant lipase (Kumari et al., 2007; Camilo Naranjo et al., 2010).
7.3.3.6. Inhibition by glycerol
Glycerol also has an inhibitory effect on lipase activity. Glycerol, being one of the products of lipase-catalyzed transesterification reaction, drives reaction equilibrium in the reverse direction. Also, glycerol molecules form a hydrophilic environment around the immobilized lipase molecule, thus preventing the hydrophobic substrate from coming into contact with the enzyme (Szczesna-Antczak et al., 2009). The continuous removal of glycerol from the reaction mixture and use of solvents are the solutions to minimize glycerol inhibition (Fjerbaek et al., 2009). Polar solvents like tert-butanol and novel solvents like ionic liquids dissolve glycerol and thus minimize its negative effect. Lipases show good stability and improved yield in such solvent systems (Gog et al., 2012). In lipase-catalyzed transesterification, acyl acceptors other than short chain alcohols, which do not lead to glycerol formation, have recently gathered interest.
7.3.3.7. Pretreatment for improving lipase stability
The stability and activity of lipases can be improved by the pretreatment of an enzyme prior to its application. The pretreatment strategy involves exposure of an enzyme to substrate and its analogs, organic solvents, and salts. These pretreatments enhance catalytic performance by keeping the active sites in open conformation. Methanol inactivation and high price are major drawbacks for lipases in their successful use for biodiesel production. Pretreatments improve the catalytic performance, methanol tolerance, as well as stability of lipases (Guldhe et al., 2015).
7.4. New tendencies in enzymatic production of biodiesel
To reduce the production cost of enzymatic transesterification, several strategies can be tried in up-, mid-, and downstream processing as proposed by Zhao et al. (2015) as shown in Fig. 7.6. Specifically, in upstream processing, the catalytic stability and activity of lipase can be improved by protein engineering, strain optimization, and metabolic engineering techniques (Hwang et al., 2014). In addition, further reduction of the running cost of the enzyme-catalyzed biodiesel production can be achieved by process-intensification strategies, for example, by improving the immobilization as well as process design and optimization. Immobilization of lipase enzymes has been studied for many years, and various carriers have been used. However, only a few types of carrier and immobilization processes have been commercialized.
Figure 7.6 Unit operations and corresponding works that can be done to reduce the cost of lipase-catalyzed biodiesel production (Zhao et al., 2015).
Nevertheless, these commercialized ILs are still too expensive to be used for biodiesel production. Some newly developed immobilization technologies by using magnetic and nano-particles have been reported, but they are still far away from industrial application. One of the solutions to the high cost of lipase for biodiesel production is to increase its life time in transesterification. At this point, reaction media, operation parameters, as well as reactor development should be considered. For example, the stability of ILs in conventional aqueous system is usually poor due to the leaching of enzyme from carriers and the inhibitive effects from methanol and glycerol (Du et al., 2008).
Reactor design is important for the scale-up of IL-catalyzed production of biodiesel, but the development of high-efficiency reactor for IL-catalyzed production of biodiesel goes slowly. Commonly used reactors are stirred tank reactor (STR), packed-bed reactor (PBR), or a combination of both. However, further improvement is still needed for intensifying mass transfer with minimizing mechanic shearing force to avoid damaging carriers and enzymes. Downstream processing is crucial to obtain biodiesel product that meets corresponding quality standards. Simulation is usually used to obtain mass and energy balance data and process optimization.
Enzymatic catalysis for biodiesel production is relatively a new research field. However, it is attracting a lot of focus from scientific community and biodiesel industry. In recent past, novel techniques have been developed to improve the sustainability and economical viability of the enzyme catalysis. These techniques mainly deal with reducing the price of enzymes as well as with improving the efficiency of transesterification conversion. In this respect, Table 7.2 shows various novel techniques and recent trends in enzymatic biodiesel synthesis and their advantages and challenges.
7.4.1. Novel immobilization techniques
Novel immobilization techniques are being developed to enhance the performance of immobilized lipase, solvent tolerance, reusability, stability, and to make the separation process easier. Protein-coated microcrystals (PCMC), cross-linked protein coated microcrystals (CL-PCMC), magnetic particle carriers, and electro-spun nanofibers are the main novel techniques for immobilization of lipases, which have been studied in biodiesel production. Enzymes, after being immobilized on magnetic particles, have the advantage of an easier separation, as well as that they become immobilized lipases that can be concentrated at specific places in a reactor by applying external magnetic fields (Dussan et al., 2010).
7.4.2. Use of lipases from different sources in combination
Lipases from different sources have shown different substrate specificity and catalytic activity. Lipases with narrow specificity are not suitable for biodiesel production. The performance of regiospecific lipases can possibly improve when used with nonspecific lipases in combination. Also, some lipases show more hydrolytic activity while others show more synthetic activity. Such lipases when used in combination enhanced the yield as well as reduced the times of reaction (Li et al., 2010; Tongboriboon et al., 2010). A wide range of feedstocks is used for biodiesel production, which is comprised of triglycerides, FFA, and regioisomers of mono- and diglycerides. The combination of lipases with distinct specificity and catalytic efficiency, when used for transesterification of such feedstocks, has shown an improved performance (Rodrigues et al., 2011). However, the preparation of such enzyme cocktails, or the development of a micro-organism expressing different lipases, via genetic engineering route, could be a very tedious process.
Table 7.2
Various novel techniques and recent trends in enzymatic biodiesel synthesis and their advantages and challenges
| Novel techniques | Advantage | Challenges |
| Use of lipases from different sources in combination | Wide substrate specificity, enhanced yields, reduced reaction time | Preparation of enzyme cocktail or genetic engineering is tedious process |
| Ionic liquids as solvent | Improved stability, selectivity, and activity of enzyme | Expensive technique |
| Enzyme-catalyzed transesterification under supercritical CO2 medium | Improves diffusion and reaction rate, salvation ability can be engineered, can be used in extraction of lipids as well, easy separation from products | Expensive technique, requires sophisticated instrumentation |
| Enzyme-catalyzed transesterification for low cost and high free-fatty-acid feedstocks | Reduces the feedstock cost, waste management by biodiesel production | Meticulous collection and logistics issues |
| Solvent-free process | Cost-effective, environmentally friendly, safe | Mass transfer limitations in reaction |
| In-situ transesterification of microalgae | Reduces solvent use, less energy consumption | Cost-effective only when the biomass has high percentage of lipids |
7.4.3. Ionic liquids as solvent in enzyme-catalyzed transesterification
The use of volatile, toxic, flammable solvents is neither safe nor environmentally- convenient. Novel solvents like ionic liquids are considered as green solvents because of their nonflammability, low vapor pressure, and high thermal stability. Ionic liquids are composed of anions and cations, which can be altered to design a suitable solvent in terms of their melting point, viscosity, density, hydrophobicity, and polarity (de los Rios et al., 2011; Zhao et al., 2013). Enzymes show higher stability, selectivity, and improved activity in ionic liquids at room temperature. Thus, ionic liquids currently are gaining interest in enzyme-catalyzed transesterification.
Hydrophobic ionic liquids have shown higher yields than hydrophilic ones. At present, ionic liquids are expensive, although they can be recovered and reused (Ha et al., 2007). Consequently, more simple recovery techniques and cheaper ionic liquids have to be investigated to understand that ionic liquid-assisted biodiesel synthesis could be economically feasible.
7.4.4. Enzyme-catalyzed transesterification under supercritical CO2 medium
To avoid the mass transfer limitations, organic solvents are being used extensively in enzyme-catalyzed biodiesel synthesis. As most of these organic solvents are toxic, volatile and flammable use of supercritical fluids as the reaction medium has gained global interest. Enzyme catalysis can be carried out in supercritical CO2 (SC-CO2) because of its moderate critical temperature and pressure, 31.1°C and 7.38 Mpa, respectively (Rathore and Madras, 2007). Supercritical CO2 as the reaction medium in lipase-catalyzed reactions offers the advantage of easy separation by reducing the pressure; also, its salvation ability can be altered by controlling temperature and pressure. Also, supercritical CO2 has been simultaneously utilized for the extraction of lipids, as well as for the solvent where the transesterification process is developed. This at some extent lowers the cost attributed to the reaction process in supercritical conditions (Taher et al., 2011).
7.4.5. Statistical approaches for optimization of reaction
Lipase-catalyzed biodiesel production is influenced by number of factors such as temperature, methanol to oil molar ratio, enzyme concentration, water content, flow rate, in case of continuous process, and so on. Thus, optimization of these parameters becomes crucial to obtain maximum yields. Statistical methods such as response surface methodology (RSM) have been widely used for the optimization of lipase-catalyzed biodiesel production (Verdugo et al., 2011; Luna et al., 2014b). Statistical methods give the advantage of studying a great number of parameters in fewer experimental setups. These methods also give a better understanding of interactions of the parameters as well as extent of on their influence on the reaction.
7.4.6. Enzyme-catalyzed transesterification for low-cost and high free-fatty-acid feedstocks
Feedstock contributes for a major portion of biodiesel production cost. Currently, edible oils are mostly used as feedstock for biodiesel production. Edible and nonedible oil crops, however, compete with food crops for arable land, which leads to food security concern. A large amount of water and fertilizers are used to grow these oil crops, which increases the cost of biodiesel production and carbon debt. Therefore, the use of low cost of waste cooking oil and animal-derived fats are gaining interest to be used as feedstocks. Besides, the use of waste cooking oil gets a dual purpose: the biofuel production and the waste management. The used cooking oil provides a cheap source of feedstock; however, for its availability at large-scale production of biodiesel, it is necessary get a very meticulous collection and logistics of this feedstock from sources like restaurants and food processing. Besides, because of waste cooking oil oxidation, it exhibits a high free-fatty-acid content (Chen et al., 2009).
Many nonedible oils, microalgal oils (Chisti, 2007; Mutanda et al., 2011a,b) such as the waste cooking oils, are known to have high free-fatty-acid and/or moisture contents. Both parameters, high FFA and high moisture contents of feedstocks, hamper the biodiesel yield when it is applied the chemical catalysis, while lipase has shown good tolerance toward these factors (Hama and Kondo, 2013). Thus, despite the high cost of enzymes, their application in converting low-quality feedstocks can improve the economic balance in the overall biodiesel production process.
The animal-derived products are usually by-products of slaughter houses and meat-processing industries. Higher calorific values and cetane numbers are the main attractive features of biodiesel derived from animal fats.
7.5. Biofuels similar to biodiesel produced using several acyl acceptors, different to methanol
To avoid the associated problems with the generation of glycerol in the conventional process, a series of alternative methods are currently considered to get higher atom efficiency. In this respect, currently the production is studied in only one reaction, of new biofuels that integrate the glycerol as a derivative product, miscible with the fatty acid methyl or ethyl esters (FAME or FAEE) obtained in the same transesterification process. This is possible by using some acyl acceptors (basically some esters), instead of the alcohol usually employed in the conventional process. In this way, in the interesterification process, the corresponding glycerol ester is obtained together the FAME (or FAEE). The mix of reaction products is constituted by lipophilic compounds completely miscible with fossil fuels, so that in that reaction is obtained a new biofuel avoiding the presence of free glycerol, which is a dangerous compound for engines, and substituted by a derivative that operates like a fuel.
Thus, this methodology avoids the separation of glycerol before its transformation, simplifying the process (Borges and Diaz, 2012; Mota et al., 2010). These biodiesel production methods not only prevent the generation of waste, but also increase the yields of the process, always higher than normal 12 wt%, by incorporating some derivatives of glycerol into the reaction products as well as all the reactants used. In this way, the highest atom efficiency, practically 100 wt%, is obtained, because every atom of reactive practically is incorporated in the reaction product. Novel methodologies to prepare esters from lipids using different acyl acceptors which directly afford alternative co-products are currently under development (Adamczak et al., 2009; Vasudevan and Briggs, 2008; Ganesan et al., 2009).
The interesterification processes can be performed with the same catalysts applied in transesterification processes (eg, homogeneous or heterogeneous, acid or basic catalysts, lipases, supercritical conditions). Although at present most of these processes, when applied to the biofuels production, are carried out using different lipases (Adamczak et al., 2009; Borges and Diaz, 2012). Instead of using methanol, the lipase-catalyzed synthesis of fatty acid alkyl esters can also be performed using alternative alcohol donors. In this respect, methyl (or ethyl) acetate as well as dimethyl (or diethyl) carbonate can be used. These mixtures, including glycerol derivative molecules, have relevant physical properties to be employed as novel biofuels. In some cases, even the unused reactants are capable of being directly used as biofuels.
7.5.1. Biodiesel produced together to glycerol triacetate in the same transesterification process of oils and fats
Mixtures of fatty acids methyl esters (FAME) and glycerol triacetate (triacetin) are products of the interesterification reaction of triglycerides with methyl acetate in the presence of strong acid catalysts (Fig. 7.7). All of these products generated from the above process could be used as components of a patented novel biofuel, which strongly improves economy of the biofuel production (Calero et al., 2015). Such a mixture, named Gliperol, has claimed that it exhibits fuel characteristics comparable with traditional biodiesel fuel (Kijenski et al., 2007). This is composed of a mixture of three molecules of FAME and one molecule of triacetin, and it can be obtained after the interesterification of one mol of triglycerides (TG) with 3 mol of methyl acetate, by using an enzymatic catalyst. A molar ratio oil/methyl acetate in the range 1:3 to 1:9, and temperatures in the range 40–200°C are usually applied. Most studies described in these processes apply lipases as catalysts, in solvent free systems (Demirbas, 2008; Usai et al., 2010), ionic liquids (Ruzich and Bassi, 2010), supercritical conditions (Saka and Isayama, 2009; Tan et al., 2010a), or ultrasound assisted interesterification (Maddikeri et al., 2013).
Figure 7.7 Gliperols is a novel biofuel proprietary by the Research Institute of Industrial Chemistry Varsow (Poland), formed by a mixture of 3 mol of FAME and 1 mol of triacetin, and obtained by interesterification of triglycerides with methylacetate under strong acidic conditions (Calero et al., 2015).
Despite the greener character of ethyl acetate, this acyl acceptor is less studied than methyl acetate (Adamczak et al., 2009; Jeong and Park, 2010; Modi et al., 2007; Kim et al., 2007), although results described indicate similar behavior to methyl acetate in the interesterification with lipases. However, in this case, the corresponding FAEE (instead of FAME) with triacetin are obtained.
With respect to the influence of triacetin on engine performance, there are a high number of studies because this molecule is considered as a good solution for the upgrading of residual glycerol obtained in the conventional synthesis of biodiesel (Rahmat et al., 2010; Melero et al., 2010). It is noted that triacetin is an antiknocking additive when it is used along with the biodiesel in DI-diesel engine, improving the performance and reducing tail pipe emissions (Casas et al., 2010). In this respect, it can be concluded that the interesterification of triglycerides with methyl or ethyl acetate may be an adequate methodology to obtain conventional biodiesel (FAME or FAEE), also including some amount of a well-recognized additive such as triacetin.
7.5.2. Biodiesel produced together to fatty acid glycerol carbonate esters in the same transesterification process of oils and fats
To this purpose, dimethyl carbonate (DMC) can be used as a transesterification reagent for making esters from lipids, which directly achieves alternative soluble co-products in the biodiesel solutions. The reaction is rather attractive, as DMC is reputed to be the prototype of green reagents due to its health and environmental inertness (Li et al., 2005). Therefore, a fuel produced using DMC and vegetable oils or animal fats as raw materials must be considered as an alternative fuel fully derived from renewable resources. Thus, DMC operates as an alternative acyl acceptor, which is neutral, cheap, and nontoxic.
The reaction between triglycerides and DMC produces a mixture of FAME and cyclic fatty acid glycerol carbonate esters (FAGCs), which constitutes a novel biodiesel-like material, named DMC-BioD (Fabbri et al., 2007) in the corresponding patent. The interesterification reaction of triglycerides with DMC can generate a mixture of FAME, FAGCs molecules, and also glycerol carbonate (GC), as it is indicated in Fig. 7.8, (Calero et al., 2015). These mixtures, including glycerol derivative molecules, have relevant physical properties to be employed as a new patented biofuel where the atom efficiency is also improved, as the total number of atoms involved in the reaction is part of the final mixture.
DMC is reputed to be a prototype of green reagents for its health and environmental inertness (Li et al., 2005), and avoided the co-production of glycerol. The main difference between DMC-BioD and biodiesel produced from vegetable oil and methanol (MeOH-biodiesel) is the presence of fatty acid glycerol carbonate monoesters (FAGCs) in addition to FAMEs. In this respect, details regarding the composition of DMC-BioD, as well as physical and rheological properties relevant for its use as a fuel, also have been studied to some extent. (Calero et al., 2015).
Figure 7.8 DMC-BioDs is a new biodiesel-like biofuel proprietary by Polimeri Europa (Italy), obtained by reacting oils with DMC under alkaline conditions, which avoids the co-production of glycerol by obtaining a mixture of 2 mol of FAME and 1 mol of FAGC. This latter can be decomposed, in this way generating GDC and GC in a variable extension (Calero et al., 2015).
In summary, with respect to benefits and drawbacks of DMC as an alternative reagent for carrying out interesterification of oil and fats to produce biofuel from renewable resources and alternative co-products (GC and glycerol dicarbonate (GDC)), it should be mentioned that DMC is a less toxic chemical than methanol that can be currently manufactured by environmentally safe industrial methods, from CO2 and renewable resources. Besides, GC and its derivatives are characterized by low toxicity, and the remaining nonreacted DMC does not need to be separated from the reaction products, because it is an effective additive for diesel engines, due to its high oxygen content (Rounce et al., 2010). Here we have that the fabrication process is very simplified with respect to the conventional biodiesel obtained from methanol.
7.5.3. Biodiesel produced together to monoacylglycerol in the same transesterification process of oils and fats
In this respect, a protocol was recently developed for the preparation of a new kind of biodiesel that integrates glycerol into their composition via 1,3-regiospecific enzymatic transesterification of sunflower oil using free (Caballero et al., 2009; Verdugo et al., 2010) and immobilized (Luna et al., 2012, 2013) pig pancreatic lipase (PPL).
Thus, the already patented Ecodiesel-100 (Luna et al., 2014c), is a mixture of two parts of FAEE and one part of MG that integrates the glycerol as a soluble derivative product (MG) in the diesel fuel, but unlike these methods, no specific reagent (such as DMC or methyl acetate) more expensive than ethanol is used. The procedure takes advantage of the 1,3 selective nature of lipases, which allows it to “detain” the process in the second step of the alcoholysis, there by obtaining a mixture of 2 mol of FAEE and one of MG, as products shown in Fig. 7.9. This strategy is based on obtaining an incomplete alcoholysis by application of 1,3-selective lipases, so that the glycerol remains in the form of monoglyceride, which avoids the production of glycerol as by-product, reducing the environmental impact of the process.
Figure 7.9 Ecodiesel-100 is a biofuel obtained by enzymatic technology patented by the University of Cordoba (UCO) incorporating glycerin, as it is formed of 2 mol of ethyl esters of fatty acids (FAEE) and 1 mol of monoglyceride (MG).
Ecodiesel exhibits similar physicochemical properties to those of conventional biodiesel. Last, but not least, monoacylglycerides (MG) were proven to enhance lubricity of biodiesel as it was demonstrated by recent studies (Wadumesthrige et al., 2009; Xu et al., 2010; Haseeb et al., 2010). Besides, ethanol does not spent in the enzymatic process remain in the reaction mixture in such a way that after the reaction the products blend obtained can be directly used as a fuel. In this respect, some studies (Cheenkachorn and Fungtammasan, 2009; Jaganjac et al., 2012) have proven that blends of diesel fuel and ethanol with biodiesel led to a slight decrease in maximum power output, with respect to regular diesel. Besides, no significant difference in the emissions of CO2, CO, and NOx between regular diesel and biodiesel, ethanol and diesel blends was observed. However, the use of these blends resulted in a reduction of particulate matter. Consequently, such blends can be used in a diesel engine without any modification, taking into account the limited changes obtained respect to the use of pure diesel. Thus, the Ecodiesel expression is currently ascribed to which ever blend of fatty acid alkyl ester is with the ethanol, alone or with any proportion of diesel fuel (Pang et al., 2008).
The Ecodiesel production with different lipases and several biocatalytic systems, as well as the main reaction parameters, have been studied, and the obtained results are summarized in Table 7.3.
Table 7.4 shows a summary sheet of the pros and cons of different existing methodologies for obtaining biofuels by integrating glycerol as a derivative. This enables them to work as combustible, together with FAME or FAEE, thus avoiding the presence of free glycerin.
In this respect, the production process of biodiesel-like biofuels by interesterification of vegetable oils with methyl acetate or methyl carbonate, used as acyl acceptors, is clearly more simple than the conventional biodiesel production, as is shown in Fig. 7.10, regardless of the use of chemical catalysis or enzymes. However, biofuels obtained by selective ethanolysis of vegetable oils using lipases as biocatalyst are even simpler, as shown in Figs. 7.10 and 7.11.
Table 7.3
Different enzymatic systems studied for the biocatalytic production of Ecodiesel
| Biocatalyst (Lipase) | Form of use | Crucial reaction parameters | References | |||
| Anova | OVAT | |||||
| Oil/EtOH | T °C | Biocatalyst weight (g) | Reuses | |||
| PPL (commercial pig pancreatic lipase) | Free | 1:2.6 | 45 | 0.01 | 0 | Verdugo et al. (2010) |
| Physical adsortion | 1:10.3 | 45 | 0.5/0.01 | 11 | Caballero et al. (2009) | |
| Covalent Inmob1 | 1:4 | 40 | 0.5/0.01 | 40 | Luna et al. (2013) | |
| Covalent Inmob2 | 1:4 | 40 | 0.5/0.01 | 25 | ||
| Lipopan (Thermomyces lanuginosus) | Free | 1:3.5 | 20 | 0.02 | 0 | Verdugo et al. (2011) |
| MML (Rhizomucor miehei) | Free | 1:6 | 30 | 0.015 | 0 | – |
| Lipozyme RM IM (Rhizomucor miehei) | Commercial immobilized | 1:6 | 40 | 0.04 | 12 | Calero et al. (2014) |
| Biolipase-R (Rhizopus oryzae) | Free | 1:6 | 20 | 0.02 | 0 | Luna et al. (2014b) |
| Physical adsortion | 1:6 | 30 | 0.5/0.01 | 9 | Luna et al. (2014a) | |
| Covalent Inmob1 | 1:6 | 30 | 0.5/0.01 | - | ||
| Covalent Inmob2 | 1:6 | 30 | 0.5/0.01 | 9 | ||
| Commercial lipases | Biocatalyst (Lipase) | Origin | Form | Crucial Reaction parameters | ||||||
| ANOVA | OVAT | |||||||||
| Oil/EtOH | T (°C) | Biocatalyst weight (g) | Reuses | |||||||
| CALB | Candida antarctica | Free | 1:6 | 30 | 0.02 | 0 | ||||
| Physical adsortion | MS 3030 | 1:6 | 30 | 0.05 | 10 | |||||
| PMO | 0.1 | |||||||||
| N435© | Commercial immobilized | 1:6 | 30 | 0.5 | 16 | |||||
| Enzymatic extracts | Wild strains | Oil environment | G. Terribacillus | Free | 1:6 | 30 | 0.5 | 10 | ||
| Animal fat environment | G. Bacillus | Free | 1:6 | 30 | 0.5 | 10 | ||||
| Standard strain | CALB (CECT) | C. antarctica | Free | 1:6 | 30 | 0.5 | 10 | |||
Table 7.4
Schematic comparison of the main characteristics of the different technologies available to produce renewable liquid fuels from vegetable oils
| Type | Biodiesel EN 14214 | Biodiesel-like biofuels | ||
| Name | Biodiesel | Gliperol | DMC-Biod | Ecodiesel |
| Reactive | Methanol or ethanol | Methyl acetate | Methyl carbonate | Ethanol |
| Catalyst | NaOH or KOH | Acid, basic or lipases | Basic or lipases | Lipases |
| Products | 3 FAME or 3 FAEE | Glycerol triacetate + 3 FAME | Fatty acid glycerol Carbonate + 2 FAME | Monoglycerides + 2 FAEE |
| By-products | Glycerol | No waste | No waste | No waste |
| Separation process and cleaning | Complex | Not needed | Not needed | Not needed |
| Investment facilities | Medium | Low | Low | Low |
| Free fatty acids and/or water in the starting oil | Free fatty acids are transformed to soaps | Free fatty acids are transformed to biofuel | Free fatty acids are transformed to biofuel | Free fatty acids are transformed to biofuel |
| Catalyst cost | Low | High | High | High |
| Environmental impact | High. Alkaline and saline effluents are generated. Wastewater treatment is needed | Low | Low | Low |
7.6. Industrial biodiesel production using enzymes
Most of the IL-catalyzed biodiesel productions in lab scale are batch reactions performed in stirred flasks, but for a larger-scale operation, the reactor must be specially designed. Several types of reactors have been studied for industrial biodiesel production, such as stirred tank reactor (STR) (Keng et al., 2008), packed-bed reactor (PBR) (Halim et al., 2009), fluidized bed reactor (FBR) (Ricca et al., 2009) and bubble column reactor (BCR) (Hilterhaus et al., 2008). However, only a few of these reactors are actually suitable for the industrial enzymatic production of biodiesel.
In order to reduce operational costs, enzymatic biodiesel must be produced in continuously operated plants. Several possible solutions obtained in laboratory scale could be CSTRs, PBRs, fluid beds, expanding bed, recirculation, or membrane reactors (Zhao et al., 2015). PBRs are very applicable for continuous biodiesel production, but the main disadvantage is that the resulted glycerol remains at the bottom of the reactor, so that this glycerol could be deposited on the surface of the support immobilized lipase, thus decreasing the catalytic efficiency. Thus, the glycerol must be continuously eliminated in a timely manner during the enzymatic reaction process. Several studies reported the successful application of PBRs for enzymatic biodiesel production using different setups: a single PBR used with stepwise addition of methanol (Zhao et al., 2015), a single recirculating PBR (Mireille Alloue et al., 2008), three PBRs in series with intermediate glycerol removal and methanol addition (Zhao et al., 2012), and nine PBRs in series with a hydrocyclone set after PBR to separate glycerol (Cheirsilp et al., 2008).
Although many processes have been developed for immobilization of lipases in lab scale, only a few techniques have been successfully commercialized. In this respect, the major drawback for the technical transfer is the high cost of lipase immobilization steps. This explain that, the market price of Novozyms 435, one of the most usual supported lipase systems, reaches to $1000/kg (Zhao et al., 2015). The immobilization process should be enough efficient for recovering proteins as much as possible, but still retaining their enzymatic activities. Besides, the ILs obtained should have high stability to avoid enzyme leaching or activity loss.
The first industrial plant for enzymatic production of biodiesel was built in China in 2006, with a capacity of 20,000 tons/year. Tert-butanol was selected as the reaction medium, and immobilized lipases like Lipozymes TL IM and Novozyms 435 were both used in this plant as enzymatic catalysts (Zhao et al., 2015).
Techno–economic evaluation is vitally important to estimate the production cost and to determine the costliest units for further optimization. Economic evaluation usually consists of several steps: the development of process flow sheets, time charts, equipment lists followed by estimations of equipment cost, and plant and manufacturing cost (Alves et al., 2013).
The economic feasibility of enzymatic production of biodiesel depends on a series of factors. These factors mainly include (1) the raw material costs such as the prices of oil feedstock, alcohol and enzyme; (2) the process parameters, such as oil-to-biodiesel conversion ratio, retention time for transesterification, biodiesel recovery yield, lipase life time, and solvent loss (if used); (3) process design regarding water recycle and heat integration; and (IV) by-product credit. It has been found that lipase cost contributes a great part of the total production cost.
The extensively used IL, Novozym 435, has a high price per kilogram, indicating that a very high productivity is required for the process to be cost-effective (Nielsen and Rancke-Madsen, 2011). Therefore, the reusability of ILs is important to reduce biodiesel production cost. As shown in Fig. 7.12, the reuse time of IL has a significant influence on enzyme cost for IL-catalyzed production of biodiesel. It can be estimated that to make the enzyme cost less than 0.1 $/kg of biodiesel, the IL should be reused for more than 320, 210, 160, 50, and 20 batches without loss of enzyme activity when lipase price are 1500, 1000, 750, 200, and 100 USD/kg, respectively.
Figure 7.12 Effect of IL reused time on the estimated lipase cost under different enzyme prices. IL loading: 2% based on raw oil feedstock; oil-to-biodiesel conversion: 95% (Zhao et al., 2015).
Techno–economic and life cycle analyses are very important for giving directions to this technology for its successful commercial-scale implementation. However, there are very few studies available on this topic. Also, it becomes imperative to compare alternative technology with the conventional technique. Jegannathan et al. (2011) investigated the economics of biodiesel production process using alkali catalyst, free, and immobilized enzyme catalysts. A production capacity of 103 tons and batch process were considered for the study. The lowest biodiesel production cost was found to be 1166.67 USD/ton for alkali catalyst. Among the biocatalyst, immobilized enzyme has shown a lower biodiesel production cost of 2414.63 USD/ton compared to free enzyme (7821.37 USD/ton). The conventional alkali catalyst price was much lower than the enzyme catalysts. Among biocatalyst, immobilized enzyme showed a lower price because of its reuse potential.
Life-cycle analysis study by Harding et al. (2008), compared the chemical catalysis and enzyme catalysis for biodiesel production. Study showed that the biological route has an advantage over the chemical route in terms of simplified purification process and energy savings. Life cycle analysis also showed that the biocatalytic route is more environmentally friendly. Global warming, acidification, and photochemical oxidation in the case of enzyme catalysis were reduced by 5%. Reduction in fresh water aquatic toxicity was approximately 12%, while reduction in marine aquatic toxicity and human toxicity were almost 10%. Reduction in terrestrial ecotoxicity was over 40%; this was mainly due to avoiding the neutralization step, which requires acids. Authors suggested these results are mainly due to lower steam requirement for enzymatic process. Even though the cost of the enzyme catalysis is higher it provides environmental benefits over the conventional process. With the last novel strategies, enzyme price can be cut down by improving its catalytic performance and stability. Both techno–economic and life cycle analysis suggest the promising potential of enzyme catalysis for biodiesel production at commercial-scale production plants.
7.7. Conclusions
Numerous lipases have been applied for biodiesel production, with a large variety of triglyceride substrates and acyl acceptors. They have been successfully used for the conversion of waste fats and oils, eliminating the main issue of traditional alkaline transesterification. However, some precaution must be taken when using methanol in order to avoid lipase inhibition. The results obtained have proved that high productivity, involving yield and numbers of reuse, as well as low reaction time, can be achieved when using enzymes. Further improvements can make industrial enzymatic biodiesel production a viable option for the future.
Lipase-catalyzed production of biodiesel has attracted great attention recently, due to the merits such as mild reaction conditions, environmental friendliness, and wide adaptability for feedstocks. Immobilization of lipase facilitates enzyme recovery and increases the stability of the enzyme. This technique shows great potential for industrial-scale production of biodiesel. Various approaches have been developed for lipase immobilization, mainly including physical adsorption, ionic bonding, covalent bonding, entrapment, and cross-linking. Nevertheless, only a few of these techniques seem to be economically feasible. Each immobilization technique has its own advantage and disadvantage, and lipase immobilization is usually performed by a combination of two or more of these approaches. Most of the commercial ILs are prepared by adsorption of free lipase on polymeric materials, because this this process is simple and the carrier is relatively easy to obtain at a cheap cost. However, the stability of ILs still should be enhanced, especially to strengthen the interaction between lipase and carriers to prevent the enzyme leaching.
On the other hand, the cost of the lipase continues to be the main obstacle for exploiting its potential. Lipase reuse is therefore essential. This can be achieved by using immobilized lipases. The industrial usage of immobilized lipases requires different qualities and characteristics of the biocatalyst depending on the specific application. Therefore, a continued effort within immobilization technology is necessary to provide solutions for each application.
Several operation parameters have been found that affect the biodiesel yield and stability of ILs. These parameters mainly include acyl acceptor types and concentration, water content, enzyme loading, alcohol to oil ratio, temperature, and reaction media. Parameter optimization is important to obtain high biodiesel yield and maximize the reuse of the enzyme. However, the optimum condition is greatly dependent on oil feedstock and the IL that is employed.
Techno–economic evaluation is important for IL-catalyzed production of biodiesel. Lipase cost contributes a significant part of the total production cost. This expenditure can be decreased by reducing the lipase loading (increasing lipase specific activity) or increasing the reusability of IL. However, to further reduce production cost, the whole process optimization with consideration of water and heat integrations should be performed.
In summary, it is mandatory that the following issues suggested be considered to improve the economic competiveness of IL-catalyzed production of biodiesel in the near future:
1. Increase the stability of ILs during transesterification: This can be done by preventing lipase from leaching off the carriers and denaturing to loss activity caused by the accumulation of alcohol and/or glycerol or shearing force of stirring.
2. Process integration and optimization should be further investigated. Since the total production cost is dependent on the whole process, the process integration with consideration of water and heat recycle should be conducted. Optimization of the whole process should be done with the production cost as the final objective function.
3. The development and maturation of new technologies to avoid glycerol generation as by-product. In this way, biofuels produced is applicable to diesel engines in a similar way that biodiesel, but without generating unwieldy waste glycerin, avoiding in this way any cleaning process with an additional high cost in water and energy.
4. Obtain more economical enzyme preparations.
Acknowledgements
Grants from the Spanish Ministry of Economy and Competitiveness, Project ENE 2011–27017 and ENE2015-70210-R, FEDER funds and Junta de Andalucía FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully acknowledged by the authors.
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