Appl Microbiol Biotechnol (2010) 85:1713–1733 DOI 10.1007/s00253-009-2383-z
MINI-REVIEW
Fatty acid alkyl esters: perspectives for production of alternative biofuels Annika Röttig & Leonie Wenning & Daniel Bröker & Alexander Steinbüchel
Received: 16 October 2009 / Revised: 23 November 2009 / Accepted: 24 November 2009 / Published online: 22 December 2009 # Springer-Verlag 2009
Abstract The global economy heads for a severe energy crisis: whereas the energy demand is going to rise, easily accessible sources of crude oil are expected to be depleted in only 10–20 years. Since a serious decline of oil supply and an associated collapse of the economy might be reality very soon, alternative energies and also biofuels that replace fossil fuels must be established. In addition, these alternatives should not further impair the environment and climate. About 90% of the biofuel market is currently captured by bioethanol and biodiesel. Biodiesel is composed of fatty acid alkyl esters (FAAE) and can be synthesized by chemical, enzymatic, or in vivo catalysis mainly from renewable resources. Biodiesel is already established as it is compatible with the existing fuel infrastructure, non-toxic, and has superior combustion characteristics than fossil diesel; and in 2008, the global production was 12.2 million tons. The biotechnological production of FAAE from low cost and abundant feedstocks like biomass will enable an appreciable substitution of petroleum diesel. To overcome high costs for immobilized enzymes, the in vivo synthesis of FAAE using bacteria represents a promising approach. This article points to the potential of different FAAE as alternative biofuels, e.g., by comparing their fuel properties. In addition to conventional production processes, this review presents natural and genetically engineered biological systems capable of in vivo FAAE synthesis.
Annika Röttig and Leonie Wenning contributed equally to this study A. Röttig : L. Wenning : D. Bröker : A. Steinbüchel (*) Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany e-mail:
[email protected]
Keywords Biodiesel . Biofuels . Fatty acid ethyl ester . Fatty acid methyl ester . Microdiesel . Renewable resources
Introduction Comparing the forecasted energy demand and accessible resources of crude oil, it is obvious that the future energy demand cannot solely be met by fossil fuels. In 2004, the currently accessible crude oil resources were estimated to be about 171.1 billion tons. By extrapolating the current consumption of about 11.6 million tons of crude oil per day, it can be estimated that the entire resources will suffice for a rather short time period only (Shafiee and Topal 2009; Vasudevan and Briggs 2008). In a very recent analysis of the global oil depletion, the UK Energy Research Centre even concluded that a peak of conventional oil production will be reached between 2020 and 2030 because wellaccessible sources will be exhausted at this time. Without appropriate alternatives to crude oil, the global economy will suffer a dramatic collapse by reason of exploding oil prices as the demand will continuously rise (Sorrell et al. 2009). Furthermore, the massive emissions of greenhouse gasses due to the combustion of fossil resources are causing an irreversible change of the global climate (World Energy Outlook 2008). Consequently, it is inevitable to resolve the dependence to crude oil and the increasing impairment of the environment by establishing a sustainable and competitive alternative which is based on renewable and abundant feedstock like biomass (Ali and Hanna 1994; Narasimharao et al. 2007) or on other regenerative sources. At present, about 90% of the biofuel market is captured by bioethanol and biodiesel, which are already applied as gasoline or diesel substitute on a large scale and are referred to as first generation biofuels (Antoni et al. 2007). Their
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production mainly relies on either simple carbohydrates (sucrose or starch) or edible vegetable oils (palm, soybean, or rapeseed). Thus, the exploitation of these cost intensive feedstocks competes with the world food supply and is economically and ethically problematic (Rude and Schirmer 2009). Due to the low yield from oilseed crops, the current diesel demand cannot be met without a dramatic increase in cultivation areas. Further conversion of natural habitats into monocultures (e.g., palm plantations in the rain forest) diminishes biodiversity and will reduce the natural carbon sink capacity (Fortman et al. 2008; Tilman et al. 2006). Converting native ecosystems to biofuel production (e.g., initiated by fire clearing) frequently cause much greater net green house gas releases over a long period than the combustion of an energy-equivalent amount of petroleum diesel would do. To reduce net emissions of green house gasses, further reduction of the storehouses of organic carbon is the wrong way. A balanced carbon dioxide (CO2) cycle can only be achieved if waste biomass and biomass from degraded cropland are used (Fargione et al. 2008). Additionally, when regarding emissions from the necessary high agricultural input and the entire production process, the overall environmental costs of first generation biofuels exceed those of fossil fuels (Crutzen et al. 2008; Pimentel et al. 2007, 2008; Ulgiati 2001; Zah et al. 2007). Biotechnology and the use of metabolically engineered microorganisms offer great potentials to overcome these limitations by enabling the conversion of low cost and abundant lignocellulosic biomass (like crop residues) to so called second generation fuels. The latter can be produced from wastes or crops, which provide higher growth rates, better yields, and lower water, fertilizer, and pesticide requirements than the currently used crops such as corn and oilseeds. These biofuels would be more advantageous for the environment and more competitive with fossil fuels, and they are also less competing with food production for fertile acreage (Fortman et al. 2008; Tilman et al. 2006). Beyond that, fuels from locally grown and abundant plants or wastes support political independence (Antoni et al. 2007). However, when replacing fossil fuels, some general properties of the final biotechnological product are essential: it should provide a high energy density, be producible at high yields (near the stoichiometric maximum for the given biomass feed), and be compatible with the existing fuel distribution infrastructure (Fischer et al. 2008).
Survey on biotechnological products for biofuels There are many possible biotechnological products, which can be used as alternative fuels. As mentioned above, bioethanol and biodiesel are already produced for substitution of fossil fuels. Furthermore, current objects of research are other
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short-chain alcohols (butanol, propanol), biogas (methane and hydrogen), alkanes, or isoprenoides. Bioethanol represents the predominant microbially produced fuel. The most common feedstocks are sugar cane-derived sucrose and corn-derived starch and glucose, which are converted by Saccharomyces cerevisiae or Zymomonas mobilis to ethanol (Fischer et al. 2008). In 2008, the global production was 65.5 billion liters, thereof 34 billion liters in the USA and 24.6 billion liters in Brazil (Licht 2008). To date, there are ambitious efforts to employ lignocellulosic material for bioethanol production, but further cost reductions are required (Gray et al. 2006). Besides the problematic feedstock, a drawback is the water solubility of ethanol, since water can lead to materials corrosion (Fischer et al. 2008). Biobutanol is in principal more suitable than ethanol because of its higher energy content, lower water solubility, and lower vapor pressure. It can be transported through existing pipelines and can be mixed with both gasoline and diesel or can be used in pure form (Atsumi and Liao 2008; Fortman et al. 2008). Due to its similar characteristics to gasoline, it is more suitable as a direct replacement than ethanol. For example, its energy density (29.2 MJ/L) is similar to that of gasoline (32 MJ/L), whereas ethanol has a lower energy density (19.6 MJ/L). Furthermore, the research octane number of butanol (96) resembles that of gasoline (91–99); however, the research octane number of ethanol is significantly higher (129; Lee et al. 2008). Butanol fermentation occurs in many Clostridium strains, but it is much more toxic to the cells and synthesized at a lower titer (maximal 20 g/L) than ethanol. In addition, its purification by distillation requires more energy due to its high boiling point (Fortman et al. 2008). The biotechnological production of butanol with Clostridium strains is described in detail in a review by Lee et al. (Lee et al. 2008). Also, butanol as well as other short chain alcohols like isobutanol or propanol can be synthesized in genetically engineered Escherichia coli via the ketoacid pathway (2-keto acid decarboxylase from Lactococcus lactis and alcohol dehydrogenase 2 from S. cerevisiae; Atsumi and Liao 2008). The biotechnological production of biobutanol as biofuel is already promoted by several companies (e.g., BP and DuPont; Lee et al. 2008). Isoprenoid-derived hydrocarbons are synthesized either via isoprenyl pyrophosphate (IPP) or its isomer dimethylallyl pyrophophate (DMAP), that can be dimerized and polymerized to form a broad array of olefinic hydrocarbons and their alcohol derivatives (Fischer et al. 2008). IPP and DMAP overproducing strains of E. coli and S. cerevisiae have already been developed. The conversion of these compounds in fuel precursors (isoprenoid alcohols or olefins) is catalyzed by phosphatases, pyrophosphatases, and terpene synthases in only one step (Rude and Schirmer 2009). Thus,
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the isoprenoid pathway opens up new possibilities to synthesize branched-chain alcohols, alkanes, alkenes, as well as cyclic hydrocarbons (Fortman et al. 2008). To date, biosynthesis of alkanes is not well understood, but it is assumed that the pathway includes the decarbonylation of fatty aldehyde intermediates (Rude and Schirmer 2009). Another possible pathway was suggested for the alkane-synthesizing bacterium Vibrio furnissii including a novel sequential reduction of hexadecanoic acid to hexadecane catalyzed by the enzyme FahR (Park 2005). Alkanes would be a more direct replacement of diesel fuel, but the yields so far described are too low for an industrial application and more knowledge of biosynthesis pathways is needed (Rude and Schirmer 2009). Biohydrogen can be used for the generation of electrical or mechanical energy without a perceptible output of CO2 (Malhotra 2007). It can be synthesized by algal or cyanobacterial biophotolysis of water or by photosynthetic or anaerobic bacteria via fermentation of organic substrates. Furthermore, hydrogen is a common byproduct of various anaerobic bacteria fermentations, for example in biobutanol plants (Antoni et al. 2007). Biogas (methane) is already produced from plant biomass like organic household, industrial waste, or energy plants (Yadvika et al. 2004). It is produced either by thermal or biological gasification, whereas the biological process is a low-temperature process with the primary product methane (60–70%) beside CO2 (30–40%). The process can be divided into three steps each of them involving a different set of anaerobic and facultatively anaerobic microorganisms. First, the polysaccharides from biomass are hydrolyzed and fermented into mainly acetic, propionic, and butyric acid as well as other minor compounds. During subsequent acetogenesis, these products are converted into acetic acid and CO2, which represents the limiting process step. The last and pivotal step of methanogenesis is carried out by Archaea (Antoni et al. 2007). Unfortunately, the entire conversion is usually incomplete and has to be optimized. As fossil methane is widely used as energy source (it represents about 20% of the US energy supply), there already exists an infrastructure for its domestic, municipal, and industrial use (Chynoweth et al. 2001). In 2007, the European electricity energy production from biogas was 19,937 GWh, whereof 9,520 GWh were produced in Germany (EurOpserv'ER 2008). With currently about 4,400 biogas plants, Germany has a world-leading position in the biogas field (German Biogas Association 2009). Biodiesel is defined as fatty acid alkyl esters (FAAE) of short-chain alcohols and long-chain fatty acids derived from natural sources like vegetable oils or animal fats and can be used in conventional diesel engines and distributed through existing infrastructure. It can be synthesized by chemical, enzymatic, and microbial (in vivo) processes
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(Adamczak et al. 2009). FAAE play an important role in many industrial fields, but also in natural processes. Fatty acid ethyl esters (FAEE) are synthesized by eukaryotic microorganisms, insects, or mammals (Best and Laposata 2003). The associated metabolic pathways and their function are very diverse and not completely understood yet (see below). Unlike FAEE, fatty acid methyl esters (FAME) are not synthesized naturally, but are of great industrial significance. This review focuses on the potential of FAAE as an alternative biofuel. For this purpose, the fuel properties of different FAAE species are examined and compared. Furthermore, different chemical, enzymatic, and in vivo synthesis processes of FAAE are described and analyzed with regard to an industrial application and their potential to displace fossil fuels. In contrast to former reviews, in this article, the presentation of several biological systems relevant for FAAE synthesis as well as a detailed comparison of fuel properties of different FAAE species are particularly emphasized.
Fatty acid alkyl esters as alternative biofuels FAAE, which are suitable for use as biofuels, consist of a long carbon chain length acyl moiety and a short carbon chain length alkyl moiety. Figure 1 shows two examples of FAAE, methyl hexadecanoate, and ethyl hexadecanoate. FAAE are synthesized by esterification of fatty acids and short-chain alcohols or by transesterification of triacylglycerides (TAGs) with short-chain alcohols (Vasudevan and Briggs 2008, Fig. 2). The transesterification reaction also generates glycerol as a byproduct (Hoydonckx et al. 2004). In 2008, the global biodiesel production was estimated to be about 12.2 million tons, with 7.7 million tons biodiesel being produced in Europe and 2.8 million tons thereof in Germany (Emerging Markets Online 2008; European Biodiesel Board 2009). The production capacity of today's existing 276 plants in Europe is estimated to be about 20.9 million tons in 2009 (European Biodiesel Board 2009). Currently, this industrial biodiesel production bases almost exclusively on the chemical transesterification of TAGs from vegetable oils employing methanol, although other short-chain alcohols could be used as well. Methanol is preferred due to its low cost and abundant availability as
Fig. 1 Chemical structures of FAME and FAEE. a Methyl hexadecanoate and b ethyl hexadecanoate
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Fig. 2 Synthesis of FAAE. a Transesterification of TAG and short chain alcohols leading to FAAE and glycerol. b Esterification of fatty acid and short-chain alcohol leading to FAAE and water. R1–4 acyl residues, R' alcohol moiety (R'=CH3 for methanol; R' = CH2CH3 for ethanol)
absolute alcohol (Al-Zuhair 2007). In few regions, like Brazil, the fermentation of sugar cane enables a low cost production of bioethanol and, thus, an industrial production of FAEE (Haas and Foglia 2005). As a feedstock for biodiesel production, ethanol is superior to methanol because it is less toxic, highly biodegradable, and can be synthesized from renewable sources via fermentation (Albuquerque et al. 2009). Since methanol is usually derived from petroleum gas, only FAEE provide complete independence from fossil resources (Korus et al. 1993; Staat and Vallet 1994). Additionally, longer-chain alcohols like ethanol, propanol, and butanol exhibit a greater solubility towards oil due to a lower polarity. Furthermore, the resulting esters provide also other advantages over FAME (see below). Depending on climate and kind of soil, different vegetable oil crops are used in biodiesel producing countries. In Europe and in the US, the worldwide leading biodiesel producers, the most commonly used oil crops are rapeseed and soybean, respectively (European Biodiesel Board 2009; National Biodiesel Board 2009). Vegetable oils mainly consist of TAGs (90–98 wt.%) and, in smaller amounts also of monoacylglycerides (MAGs) and diacylglycerides (DAGs), free fatty acids (FFA; 1–5 wt.%), phospholipids, phosphatides, carotenes, tocopherols, as well as traces of sulfur components and water (Brown 1938; Goering et al. 1982). Depending on plant species and environmental impacts, the fatty acid composition of TAGs varies. Nevertheless, the most frequent compounds are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3) in different proportions (Goering et al. 1982; Kapur et al. 1982). Experiments concerning the utilization of vegetable oils as fuels reach back to the beginning of the twentieth century when Rudolf Diesel tested neat vegetable oils in diesel engines (Diesel 1913). Their potential to serve as alternative fuels was particularly considered in precarious situations, like World War II or the oil crisis in the 1970s
(Knothe 2001). Nevertheless, plant oils cause serious engine problems, among others, due to their high viscosity and low volatility, and they are therefore not really suitable as an alternative to diesel fuel (Graboski and McCormick 1998; Meher et al. 2004). These properties can be ascribed to the high molecular weight and chemical structure of plant oils (Demirbas 2003; Goering et al. 1982). To overcome these problems, TAGs or FFA are (trans)esterified with short-chain alcohols to biodiesel, which greatly affects the fuel properties (Vasudevan and Briggs 2008; see below). The final oil yield of common oil crops is very poor, e.g., rapeseed yields only 1,300 L/ha and oil palm about 5,950 L/ha as the oil content of common crops is less than 5% of total biomass (Chisti 2007; Kalscheuer et al. 2006). Contemplating the oil content reached by some microorganisms or microalgae (more than 80% of the cellular dry weight), it is obvious that far better resources for biodiesel production exist (Alvarez and Steinbüchel 2002; Bröker et al. 2009; Chisti 2007; Metzger and Largeau 2005). Thus, alternative oil sources must be developed to completely replace fossil fuels since not enough acreage for oil and food crops is available. At the current rate of consumption in the USA, about 530 billion liters biodiesel would be required per year. That would necessitate about 111 million hectares of palm plantations, but only about 5.4 million hectares of microalgae plants, if an average algae oil content of 30% dry weight is assumed (Chisti 2007). Further advantages of microalgae are their high growth rate, their ability to grow in brackish or sea water on land that is not suitable for agriculture, and a possible sequestration of CO2 from flue gasses emitted from industrial plants (Hu et al. 2008). Animal fats, fat waste and residuals are often recommended as further alternatives to vegetable oils. However, due to the high degree of saturation of the fatty acids in TAGs from animal, their esters have very poor cold flow properties (Ma and Hanna 1999). Since waste fats have a high content of FFA, they cannot be transesterified using
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the common processes and will need a pretreatment (see below; Vasudevan and Briggs 2008). A further, non-vegetable source of fatty acids is tall oil, a byproduct of paper pulp production. The production of one ton paper pulp results in 30–40 kg tall oil. It contains a mixture of fatty acids (42–55%), rosin acid (33–47%), sterols, and other minor components. Tall oil fatty acids mainly consist of unsaturated fatty acids like oleic (25– 45%), linoleic/linolenic acid (45–65%), and 1–3% saturated fatty acids (Zoebelein 2001). The methylation of tall oil fatty acids yielding biodiesel was subject of several studies. Tall oil was found to be a suitable alternative source for fatty acids, particularly due to the abundance of lignocellulosic biomass (Altiparmak et al. 2006; Keskin et al. 2006; Lee et al. 2006). Commercially distributed biodiesel have to meet the European (DIN EN 14214) or American standard specifications (ASTM D-6751). The most important features are viscosity, cetane number, flash point, pour point, and cloud point. High viscosities impair the transport through pipelines and injection nozzles and can lead to an insufficient combustion in the cylinder. The viscosity rises with increasing chain length and degree of saturation. In Europe, this parameter has to be between 3.5 and 5 mm²/s. An indicator for the ignition behavior of a chemical compound is its cetane number, as a high cetane number stands for a short ignition delay and thereby a high main ignition time. The flash point describes the tendency of the fuel to build a flammable mixture with air and is helpful to estimate the ignition danger of a chemical compound (Canakci and Sanli 2008; Srivastava and Prasad 2000). According to the European standard specifications, the cetane number has to be higher than 51, and the flash point must be higher than 101 °C. The pour and cloud points of biodiesel are crucial for its cold flow properties and mainly depend on degree of saturation. Below the temperature of the cloud point, wax crystals accrue which lead to a turbidity of the fuel and may cause fuel lines and filters to be plugged. The lowest temperature, at which fuel is still liquid and can be pumped through fuel lines, is denoted as the pour point (Canakci and Sanli 2008). Furthermore, the emission profile of biodiesel is subjected to restrictions, since the output of carbon monoxide (CO), particulate matter (PM), and nitrogen oxides (NOx) is not allowed to exceed legally set values.
Properties of FAAE In comparison to conventional diesel fuel, FAAE have higher cetane numbers, pour points, cloud points, and flash points as well as a greater viscosity, a higher oxygen but lower sulfur content and heat of combustion (Peterson et al.
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1995). The higher pour points and cloud points of biodiesel account for worse cold flow properties, which limit its usage in cold climate zones (Knothe 2005). Unlike diesel fuel, FAAE are denser, less compressible, and have lower energy content. The latter is due to a higher specific weight and an oxygen content of about 10 wt.%. Thus, the effective fuel consumption is increased (Peterson et al. 1995; Foglia et al. 1997). Nevertheless, the high flash points of biodiesel enable a safer handling and transport of this fuel. Furthermore, the high oxygen and low sulfur contents improve the emission profile (see below). Physical and fuel properties of FAAE mainly depend on the chemical structure of the fatty acid moiety, like chain length, degree of saturation, and branching of the chain (Knothe 2005). However, the used alcohol has an impact as well. In general, the pour and cloud points decrease with increasing chain length and branching of the alcohol moiety (Foglia et al. 1997, see Table 1). Although (iso-)butyl or (iso-)propyl esters appear to have enhanced fuel properties in comparison to the common methyl esters, their usage is limited due to the higher price of the alcohols (Knothe 2005). Hence, most studies are restricted to methyl and ethyl esters as they are more practicable alternatives to conventional diesel fuel. The following comparison focuses on the slight differences between corresponding FAME and FAEE, which are due to the alcohol moiety, and the additional carbon atom, respectively. FAEE exhibit several advantages over FAME like increased cetane number and energy content, decreased density, and pour and cloud points (Table 1). Thus, FAEE combusts to a larger extent, the effective fuel consumption is reduced, and the cold flow properties are improved (Peterson et al. 1995; Foglia et al. 1997). Due to a relation between density and emissions of NOx and PM, the usage of FAEE instead of FAME causes lower NOx emission values and smoke densities (McCormick et al. 2001; Peterson et al. 1992; see below). This is associated to a slightly reduced carbonization of the combustion chamber and injection nozzle. It has been found, that the degree of carbonization is similar to that of conventional diesel fuel (Peterson et al. 1995; Clark et al. 1984). The maximum engine performance of both FAME and FAEE is lower in comparison to that of fossil diesel fuel. In contrast to the aforementioned advantages of FAEE over FAME, this parameter diminishes more extensively in the case of FAEE as could be shown by Clark et al. (1984) or Zajac et al. (2008). For example, using FAEE, the maximum engine performance decreased by 4% whereas that of FAME was decreased by 2.5% as compared to diesel fuel (Clark et al. 1984). Emission profiles In general, the emission values of oxygenated fuels, like FAME or FAEE, are better since the combustion is enhanced and more complete (McCormick
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Table 1 Relevant properties of different FAAE, the reference diesel fuel and some vegetable oils Fuel
Viscosity [cSt]
Density [kg/m3]
Heat of combustion [MJ/kg]
Cetane number
Pour point [°C]
Cloud point [°C]
Flash point [°C]
Sulfur content [wt.%]
No. 2 Diesel
2.39 3.51a 37.3 33.1 4.08
838.3
45.8
−23
−19
37.5 37.9 46.2
−31.7 −12.2 n.d.
−3.9 −3.9 n.d.
78 80.5a 246 254 141
0.25
912 914 884
45.71 45.2a 39.7b 39.6b 39.8
4.14 4.41 4.40 5.65 6.17 2.98 5.65 6.17 3.89 4.49 4.81 5.04 3.56
887 881 882 880.2 876 849.5 n.d. n.d. n.d. n.d. n.d. n.d. 880
39.9 40 40 40.54 40.51 45.54 37.77 38 37.04 37.44 37.25 37.63 39.8
48.7 48.2 49 61.8 59.7 49.2 61.8 64.9 54.8 52.7 72.7 72.4 48.6
n.d. n.d. n.d. −15 −10 −16 −15 −10 −3 −3 16 12 −3
n.d. n.d. n.d. 0 −2 −12 n.d. n.d. n.d. n.d. n.d. n.d. −1
135 160 157 179 124 74 179 163 188 171 160 185 175
0.01 0.01 0.01 0.012 0.012 0.036 0.012 0.014 0.012 0.008 0.01 0.009 <0.01
3.43 n.d. n.d. n.d. n.d. n.d. n.d. 4.3 4.4 7.9 6.2 7.3 7.1 6.9 7.4 6.8
870 887.7 881.7 868.4 863.6 894.3 886.9 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
40 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
49.1 47.2 47.3 86.9 76.8 41.7 44.4 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
−6 n.d. n.d. n.d. n.d. n.d. n.d. −2 −4 15 12 9 0 6 3 0
−3 n.d. n.d. n.d. n.d. n.d. n.d. 0 1 17 15 12 8 9 8 9
188 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
<0.01 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Rapeseed oil Soybean oil SME SME+additives SEE SEE+additives RME REE D2 Diesel RME REE SME SEE TME TEE C. cardunculus methyl esters C. cardunculus ethyl esters SME SEE Methyl stearate (C18:0) Ethyl stearate (C18:0) Methyl linoleate (C18:2) Ethyl linoleate (C18:2) SME SEE TME TEE TPE TiPE TBE TiBE T2-BE
0.01b 0.01b 0.01
Reference
Clark et al. 1984 Peterson et al. 1992a Canakci and Sanli 2008 Srivastava and Prasad 2000b Clark et al. 1984
Clark et al. 1984
Peterson and Reece 1994
Encinar et al. 2007 McCormick et al. 2001
Foglia et al. 1997
n.d. no data, REE rapeseed ethyl esters, RME rapeseed methyl esters, SEE soy ethyl esters, SME soy methyl esters, TBE tallow butyl esters, TEE tallow ethyl esters, TiBE tallow isobutyl esters, TiPE tallow isopropyl esters, TME tallow methyl esters, TPE tallow propyl esters, T2-BT tallow 2-butyl esters
et al. 2001; Canakci and Sanli 2008). Due to the low sulfur content of biodiesel, the emission of sulfur oxides is remarkably reduced in comparison to conventional diesel fuel. Furthermore, the output of hydrocarbons (HC), CO, and PM significantly decreases, whereas the NOx emissions rise with an increasing biodiesel fraction in biodiesel-diesel blends. These relations have been verified in several studies, particularly in a large-scale analysis of the Environmental Protection Agency (EPA) in 2002 (EPA 420-P-02-001;
Mittelbach and Tritthart 1988; Marshall 1993; Wyatt et al. 2005; Canakci and Sanli 2008). Hitherto, the reasons for an increased NOx formation during combustion of biodiesel are not completely understood. It is assumed that a higher boiling point and the presence of carbon double bonds are partly responsible since the NOx emissions of FAAE rise with increasing chain length and ratio of unsaturated fatty acid moieties (McCormick et al. 2001). Moreover, several studies
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revealed an impact of the alcohol moiety. For example, the combustion of soybean oil ethyl esters produced between 1.4% and 3.4% less NOx than the corresponding methyl esters (Clark et al. 1984; Peterson and Reece 1994; McCormick et al. 2001). However, a broad examination by Peterson and Reece (1994) of rapeseed oil methyl and ethyl esters revealed another finding regarding NOx and PM. In a total of 39 tests on a chassis dynamometer, the NOx emissions of FAAE were lower and the output of PM was higher in comparison to diesel fuel. This discrepancy might be due to differing fatty acid constituents of the rapeseed oil or some features of the used particular engine, because older, lower injection pressure engines are more sensitive to an increased cetane number resulting in significant reductions of NOx output (Peterson and Reece 1994; Knothe 2005). Nevertheless, according to many other emission studies, the ethyl esters performed better than the corresponding methyl esters concerning the output of NOx, HC, CO, and PM, which was reduced on average by 3.6%, 8.5%, 3.2%, and 4%, respectively (Peterson and Reece 1994). Generally, the problem is a poor comparability of emission results for different fuels as the test records often vary. Nonetheless, until now, advantages of FAEE over FAME have been observed in many studies. Storage stability A critical aspect for quality evaluation of biodiesel is its storage stability. During storage of biodiesel, microbial, hydrolytic, or oxidative degradation may occur (Meher et al. 2004; Peterson and Möller 2005; Srivastava and Prasad 2000). Contrary to diesel, biodiesel is less toxic and readily biodegradable because a multitude of microorganisms possesses hydrolytic enzymes to cleave the ester bond (see below). In addition to microbial decomposition, chemical hydrolytic degradation may be caused and enhanced by means of water and acids, respectively. The higher the content of MAGs and DAGs, the more water can be absorbed (Srivastava and Prasad 2000). Oxidative degradation is related to the number of carbon double bonds of fatty acids, content of hydroxyperoxides or natural antioxidants, temperature, and presence of ultraviolet light or air (Srivastava and Prasad 2000). Therefore, FAAE with a high degree of saturation are more resistant to oxidation and more stable in presence of light, oxygen, high temperatures, and metals (Canakci and Sanli 2008; Knothe 2005). Oxidation of biodiesel dramatically diminishes its quality due to the formation of gum as it does not burn completely and causes carbon deposits in the combustion chamber for example (Canakci and Sanli 2008). Hence, FAAE should be stored in photoresist and airtight tanks at temperatures below 30 °C. Under these conditions, differences in the storage stability between FAME and FAEE are negligible (Du Plessis et al. 1985). Antioxidants, which
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are naturally contained or added after biodiesel production, can significantly improve the storage stability of biodiesel. Ethanolysis of cottonseed oil, which inherently contains a high gossypol concentration (about 1.5% of the cotton seeds) results in FAEE with enhanced storage stability due to the antioxidant properties of the pigment gossypol. For this purpose, methanol is not applicable as it releases the gossypol out of the oil, and thus lowering the envisaged effect (Joshi et al. 2008). A further advantage of cottonseed oil is its abundance as a byproduct of the cotton industry thereby being a low-priced feedstock that does not compete with edible crops (Joshi et al. 2008). Biodegradability Microbial degradation is advantageous if biodiesel unintentionally passes into the environment. Many studies on the environmental impact of spilled diesel or biodiesel/diesel blends and the bioremediation of contaminated areas have revealed that biodiesel is less toxic and much easier degraded than diesel fuel (Khan et al. 2007; Koo-Oshima et al. 1998; Lapinskiené et al. 2006; Makareviciene and Janulis 2003; Mariano et al. 2008; Pasqualino et al. 2006; Zhang et al. 1998). Zhang et al. (1998) postulated three main reasons for this observation. Firstly, the rate of a reaction is regulated by the amount of catalyzing enzymes in a cell, so that a rapid degradation requires sufficient available enzymes. Because ester compounds are synthesized naturally by many organisms, degrading enzymes for FAAE exist naturally. Appropriate enzymes of the β-oxidation pathway, like acyl-CoA dehydrogenase, recognize the biologically active oxygen atoms in the biodiesel molecules and attack them by oxidizing the β-carbon atom and convert the biodiesel molecule to an acetyl-CoA and an acyl-CoA molecule with two less carbon atoms. In contrast to biodiesel, diesel contains large amounts of alkanes and alkenes (from C10– C20) which do not contain oxygen atoms and are therefore not biologically active. Secondly, the composition of diesel is chemically more complex and its biodegradation necessitates more energy. At last, some diesel compounds are toxic to microorganisms contributing additionally to the worse biodegradability. However, through mutation of genes encoding for appropriate enzymes or by producing new enzymes bacteria can adapt rapidly, so some bacteria are able to degrade diesel. Pitter and Chudoba (1990) concluded that the degradation of diesel fuels starts with oxidation of a terminal CH3 group to a carboxylic group followed by β-oxidation of the built carboxylic acid. The capability of biodegradation by natural cultures is inhibited by often used artificial antioxidants like tert-butylhydroquinone (Junior et al. 2009). To solve this problem and to advance the biological breakdown of the fuel pollutant by indigenous microorganisms, they can be stimulated by modifying nutritional value, oxygenation, temperature, pH,
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and by addition of surfactants. Furthermore, adequate strains, e.g., Pseudomonas aeruginosa LBI or Candida viswanathii, which are specialized on a rapid degradation of biodiesel/diesel blends and/or tolerate this toxic additives, can be inoculated (bioaugmentation; Junior et al. 2009; Mariano et al. 2008).
Chemical synthesis processes of FAAE For the current industrial production of biodiesel, chemical catalysis is of prime importance (Lotero et al. 2005). Alkaline or acidic, homogenous or heterogenous chemical catalysts can be used. In industrial production, homogenous alkaline catalysts are prevalent because they cause less corrosion and their reaction rate is about 4,000 times higher compared to acidic catalysts (Formo 1954; Srivastava and Prasad 2000). The most common homogenous alkaline catalysts are sodium and potassium hydroxide, due to their low cost, as well as sodium methoxide and ethoxide, which are more reactive (Lotero et al. 2005; Narasimharao et al. 2007). The optimal catalyst concentration is between 0.5 and 1%, and FAAE yields between 94% and 99% can be obtained (Feuge and Gros 1949; Krisnangkura and Simamaharnnop 1992). The major drawback of alkaline catalysts is that they lead to saponification, if FFA is present in the reaction medium (Lotero et al. 2005). Water increases saponification due to hydrolysis of FAAE to additional FFA. In consequence, the feedstocks for alkaline-catalyzed biodiesel production must be anhydrous, and the FFA content has to be lower than 0.5 wt.% (Bradshaw and Meuly 1944; Feuge and Gros 1949; Haas 2004; Ma et al. 1998). These
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conditions necessitate the usage of cost-intensive anhydrous alcohols and high-quality refined vegetable oils because animal fats or waste fats normally exhibit FFA levels above 6 wt.% (Haas et al. 2005). The advantages of acidic catalysts are their lower sensitivity to FFA and their ability to catalyze the esterification of FFA. However, due to the low reaction rate, the acidic process itself is not of great importance for biodiesel production, but can be applied as pretreatment of FFAcontaining feedstock followed by alkaline catalyzed transesterification (Lotero et al. 2005; Srivastava and Prasad 2000). Furthermore, enzymatic processes can be linked to chemical transesterification to esterify remaining FFA (Haas and Scott 1996). Both alkaline and acidic catalyzed transesterification proceed in three consecutive, reversible steps (Fig. 3). In case of alkaline catalysis (Fig. 3a), the nucleophilic alkoxide attacks the carbonyl group of the TAG molecule, forming a tetrahedron intermediate, which interacts with the alcohol resulting in an alkyl ester and the regenerated alkoxide ion. The acidic catalysis starts with protonation of a carbonyl oxygen atom of the TAG molecule (Fig. 3b). The subsequent nucleophilic attack of the alcohol results in a tetrahedron intermediate, which dissociates in one DAG and one FAAE molecule (Lotero et al. 2005). The final yield of FAAE is not influenced by the type of alcohol, but longer-chain alcohols are better miscible with oil and allow higher reaction temperatures, accelerating the reaction rate (Freedman et al. 1984; Srivastava and Prasad 2000). In contrast, the conversion yield is strongly affected by the ratio of alcohol to oil. The stoichiometric alcohol/oil ratio is 3:1 (Freedman et al. 1984). To shift the equilibrium towards product site, the ideal molar ratio was reported to
Fig. 3 Mechanisms of alkaline (a) and acidic (b) catalyzed FAAE production. R1–4 acyl residues, R' alcohol moiety (R'=CH3 for methanol; R' = CH2CH3 for ethanol)
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be 6:1 for alkaline catalysis and up to 30:1 for acidic catalysis (Canakci and van Gerpen 1999; Freedman and Pryde 1982; Freedman et al. 1984). Additionally, acidic catalysis requires higher temperatures (>100 °C) than alkaline catalysis (60–70 °C). To maximize the contact surface of the reactants, mechanically mixing is essential (Srivastava and Prasad 2000). Another possibility to increase the surface is the application of ultrasonification (Armenta et al. 2007; Wu et al. 2007). Furthermore, a homogenous reaction mixture can be achieved by adding organic solvents like tetrahydrofurane (Zhou et al. 2003). Although the chemical catalysts are comparatively cheap and offer high reaction rates, this technique exhibits several drawbacks. It is energy consuming, requires a neutralization of the catalyst, as well as several final purification steps due to the complicated removal of the catalyst and glycerol. Furthermore, the industrial alkaline catalysis necessitates high quality, water and FFA free feedstocks (Fukuda et al. 2001; Nielsen et al. 2008).
Enzymatic (in vitro) synthesis of FAAE Enzymatic catalysis enables the use of less qualitative oils and fats (with higher contents of FFA) requires only low alcohol to oil ratios and mild reaction conditions and facilitates product separation, thereby allowing lower energy consumption and significantly reduced production costs (Du et al. 2008; Nielsen et al. 2008; Rodrigues et al. 2008). The main drawbacks of enzymatic catalysis are the cost-intensive preparation of enzymes and their possible inactivation through short-chain alcohols like methanol or oil components (Akoh et al. 2007). Presently, enzymatic catalysis is almost exclusively done at the laboratory scale, except one biodiesel production plant in China that produces about 20,000 tons of biodiesel per year (Du et al. 2008). Enzymatic (in vitro) synthesis of FAAE is accomplished by lipases, which are formally referred to as triacylglycerol acylhydrolases (EC 3.1.1.3). They are naturally synthesized by not only microorganisms like fungi or bacteria but also by plants and animals for hydrolysis of TAGs to FFA and glycerides. Besides, lipases also catalyze the esterification of carboxylic acids with alcohols (Nielsen et al. 2008). During the time course of their catalyzed reaction mechanism, TAGs are in general transesterified directly with short-chain alcohols (Kaieda et al. 1999; Fig. 4). Because of low production costs and an easy modification of their properties, commercially used lipases are mainly isolated from microorganisms, preferably eukaryotic, filamentous fungi-like species of the genera Thermomyces, Rhizopus, Mucor, or Candida. Some lipases are also isolated from prokaryotes, in particular from species of the genera Pseudomonas or Burkholderia (Mrozik et al. 2008).
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The reaction rate and final biodiesel yields of enzymatic catalysis depend, like in chemical catalysis, on temperature, alcohol to oil ratio, and water content of the reaction system. However, in enzymatic catalysis also the type of alcohol, the use of solvents and the properties of the used lipase influence the reaction process (Antczak et al. 2009). Since many examples have already been discussed, which concern the effect of parameters on biodiesel yield (e.g., Adamczak et al. 2009; Uthoff et al. 2009), this chapter focuses on the main principles of enzymatic catalyzed biodiesel synthesis. The first experiments in enzymatically catalyzed FAAE synthesis were carried out with free (soluble) enzymes. Meanwhile, free and immobilized lipases are used for this purpose. Although the preparation costs for free lipases are less than for immobilized lipases, the production costs for biodiesel are increased due to a poor reusability of free enzymes (Nielsen et al. 2008). In addition, they necessitate long reaction times to achieve high biodiesel yields. In consequence, most experiments concerning enzymatic catalysis focus on immobilized lipases, which can be easily recaptured and reused (Du et al. 2008). Moreover, the immobilizations has a stabilizing effect on the enzyme, and allow the application of higher reaction temperatures, which lead to a faster conversion rate and a shorter reaction time (Fjerback et al. 2009; Nielsen et al. 2008). The most often used immobilized enzymes are lipases isolated from Candida rugosa, Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozym RM IM), Thermomyces lanuginosus (Lipozym TL IM), Mucor miehei (Lipozym IM60), Rhizopus oryzae, and Pseudomonas cepacia (Du et al. 2008; Nielsen et al. 2008). In contrast to chemical catalysis, which usually proceeds at temperatures slightly below the boiling point of the alcohol (>60 °C), the optimal temperature for enzymatic catalysis depends on the used lipase and is mostly between 30 and 45 °C. It is a compromise between enzyme stability, which decreases with an increasing temperature, and transesterification rate, which increases with raising temperatures. It is also influenced by the molar alcohol to oil ratio and the type of solvent (Rodrigues et al. 2008). For enzymatic catalysis, almost all types of alcohol can be used, although short-chain alcohols like methanol readily inactivate the lipase. Because this inactivation is ascribed to the low solubility of short chain alcohols in the hydrophobic oil, and the degree of inactivation is inversely proportional to the carbon atoms in the short chain alcohol, methanol seems to be less adequate than ethanol for enzymatic catalysis (Chen and Wu 2003). Although reaction rates with longer chain alcohols are generally higher, the final FAAE yields depend on the substrate specificity of the lipase (Fukuda et al. 2001; Shimada et al. 2002). Beside the type of alcohol, the alcohol to oil ratio
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Fig. 4 Mechanism of lipase-catalyzed transesterification. 1 The histidine residue attracts the hydrogen atom of the hydroxyl group of serine building an oxygen anion. It attacks a carbonyl atom of TAG resulting in tetrahedron intermediate I. 2 The proton of histidine is transferred to the separating DAG yielding an acyl enzyme intermediate. 3 The serine ester interacts with the alcohol molecule. First, the nitrogen atom of the histidine attracts the hydrogen atom of the
alcohol building an alkyloxid anion (R-O−). This alkyl oxid anion attacks the carbonyl carbon atom of the serine ester yielding the tertrahedron intermediate II. 4 In the last step, the FAAE molecule and the free enzyme are released (Al-Zuhair 2007; Jegannathan et al. 2008). R1– R3 long-chain acyl moieties, R’ short-chain alkyl moiety of the alcohol, His histidine, Ser serine, Asp aspartic acid, Ox oxygen atom
plays a crucial role as well. Unlike in chemical catalysis, a stoichiometric ratio of alcohol to oil (3:1) suffices for high biodiesel yields in enzymatic catalysis. An excess of alcohol may lead to higher yields, but can also cause inactivation of lipases. For methanol, it has been demonstrated that C. antarctica lipase is already inactivated by methanol/oil ratios of 0.5:1 or higher (Shimada et al. 1999; Xu et al. 2003). This problem can be solved by stepwise addition of alcohol (Shimada et al. 1999, 2002; Watanabe et al. 2000, 2001). Enzymatic biodiesel synthesis can be accomplished in aqueous media, water-free organic solvents, compressed gasses, or supercritical fluids. In aqueous media, TAGs are initially cleaved (into FFA and glycerol) and the FFA is subsequently esterified (Selmi and Thomas 1998). In contrast to chemical catalysis in industry, in which the water content has to be very low, some lipases are able to catalyze the transesterification in presence of 20% water. Although an increasing water amount rather inactivates lipases, they always need a minimal water content (∼0.48 wt.%) to maintain their active three-dimensional structure (Bommarius and Riebel-Bommarius 2000; Zhao et al. 2007). Since an excess of water shifts the equilibrium towards hydrolysis thereby lowering the biodiesel yield, the
optimal water content is always a compromise between maximal enzyme activity and minimal ester hydrolysis (Noureddini et al. 2005). Organic solvents like n-hexane (Nelson et al. 1996; Soumanou and Bornscheuer 2003), petroleum ether (Du et al. 2007; Lara and Park 2004), or t-butanol (Du et al. 2007; Li et al. 2006; Royon et al. 2007; Wang et al. 2006), instead of aqueous reaction medium, increase the solubility of alcohol and allow higher alcohol concentrations (up to an alcohol/oil ratio of 6:1) without inactivation of lipases (Antczak et al. 2009). The hydrophobic organic solvents n-hexane and petroleum ether already increase enzyme activity, whereas highest yields can be achieved with the hydrophilic organic solvent t-butanol, which diminishes the problem of undissolved alcohol droplets (Du et al. 2008). Nevertheless, the use of organic solvents leads to a reduced capacity of the reaction chamber, environmental pollution, and high costs (Nielsen et al. 2008). In contrast to liquid organic solvents, supercritical fluids (like supercritical CO2) offer low viscosities and surface tenses. Hence, diffusion rates are increased and catalysis is accelerated. Moreover, only small amounts of supercritical CO2 are necessary to achieve yields comparable with organic solvents, and the supercritical fluids do not need to be
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separated from the final products. Drawbacks of supercritical fluids are lower conversion rates and strongly reduced enzyme activities (Oliveira and Oliveira 2001). In contrast to supercritical fluids, compressed gasses lead to a significant increase of enzyme activity (Habulin and Knez 2001). A method, which does not require any solvents, is the use of methyl acetate or ethyl acetate as acyl acceptors (Du et al. 2004; Kim et al. 2007; Xu et al. 2003). These compounds show an enhanced miscibility with the oil phase, avoid the inactivation of lipases by undissolved alcohol droplets, and thus increase lipase stability. Table 2 summarizes several examples regarding the impact of the named parameters on enzymatic catalysis.
Lipase-mediated synthesis of FAAE using whole-cell biocatalysts Instead of using purified lipases, whole-cell biocatalysts (WCB) can cause significantly reduced costs because isolation, purification, and immobilization of the lipase are omitted (Fukuda et al. 2008). Since the usage of WCB for biodiesel production was discussed intensively in recent reviews (e.g., Uthoff et al. 2009), only essential principles are presented here, whereas many examples for successful applications of WCB for biodiesel production are listed in Table 3. WCB like bacteria, yeast, and fungi are easily being cultivated. The filamentous fungi Rhizopus chinensis and R. oryzae are particularly suitable for industrial applications because they are robust and immobilize spontaneously on biomass support particles during cultivation (Fukuda et al. 2001). This immobilization does not only allow a repeated use of the WCB, but also increases the specific intracellular lipase activity four- to sevenfold in comparison to suspended cells (Nakashima et al. 1990). The R. chinensis lipase catalyzes the esterification of FFA very efficiently (He et al. 2008). An impressive conversion yield of 93% ethyl hexanoate was obtained after pretreating the cells of R. chinensis with yatalase and organic solvents (Wang et al. 2007). So far, many studies focused on R. oryzae (Ban et al. 2001, 2002; Hama et al. 2006; Jin et al. 2008, 2009; Li et al. 2007b). This fungus expresses two lipases, one bound to the cell wall surface and the other to the cell membrane with the membrane-bound lipase more significant for methanolysis (Hama et al. 2006). S. cerevisiae is also of great interest for WCB, as it is easily cultivated to high cell densities and as it exhibits a rigid cell wall that holds its structure in presence of organic solvents. Furthermore, successful heterologous expression of intracellular eukaryotic lipases or of cell surface display systems has been demonstrated (Matsumoto et al. 2001, 2002; Uthoff et al. 2009). The use of bacteria like E. coli or Bacillus subtilis
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as WCB for production of FAAE is relatively new and seems to be a promising alternative (Uthoff et al. 2009).
Acyltransferase-mediated in vivo synthesis of FAAE (microdiesel) The current biodiesel production is problematic as it relies almost exclusively on vegetable oils and petrochemically derived methanol. Furthermore, the high costs for feedstock (up to 70–85% of total costs) as well as for the energy consuming transesterification and purification processes render the production uneconomical (Dai et al. 2007; Kalscheuer et al. 2006). The "microdiesel" approach aims at a complete microbial synthesis of FAEE from simple and sustainable carbon sources. This promising concept was recently established for the first time in genetically engineered E. coli strains for FAEE biosynthesis (Kalscheuer et al. 2006). On the one hand, an effective ethanol synthesis pathway was established by introducing the two genes adhB (for alcohol dehydrogenase B, AdhB, EC 1.1.1.1) and pdc (for pyruvate decarboxylase, Pdc, EC 1.1.1.4) from Z. mobilis. The Gram-negative species E. coli is only able to synthesize small amounts of ethanol via mixed acid fermentation under anoxic conditions (from acetylcoenzyme A via acetaldehyde by the aldehyde/alcohol dehydrogenase). By heterologous expression of the two genes of the ethanol synthesis pathway of Z. mobilis, this amount can be significantly increased (up to 95% of all fermentation products; Ingram et al. 1987). The central intermediate pyruvate is decarboxylized by Pdc to acetaldehyde, which is reduced to ethanol via AdhB (Neale et al. 1986). On the other hand, the heterologously expressed wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) catalyzed the in vivo esterification of ethanol and the fatty acid moiety of acyl-CoA to FAEE (Kalscheuer et al. 2006). The essential enzyme for FAEE synthesis is the promiscuous acyltransferase WS/DGAT from the Gramnegative bacterium Acinetobacter baylyi strain ADP1 (formerly A. calcoaceticus strain ADP1) which plays a key role for wax ester and TAG accumulation under storage conditions (Fixter et al. 1986; Kalscheuer and Steinbüchel 2003). WS/DGAT has an extremely broad substrate range. It is capable of catalyzing the esterification of linear alcohols from C2 to C30, cyclic, aromatic alcohols, MAGs, or DAGs with saturated or unsaturated acyl-CoA thioesters with chain lengths ranging from C2 to C20. It shows the highest activity towards middle-chain length alcohols (C14– C18) and palmitoyl-CoA, whereas short-chain alcohols like ethanol or butanol are converted with a lower but detectable rate (Kalscheuer et al. 2003; Stöveken et al. 2005). Furthermore, even the formation of wax diesters, thio, and
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Table 2 Transesterification of TAGs to FAAE by isolated lipases Origin of lipase
Acyl acceptor
Oil source
Free lipases C. rugosa P. cepacia P. cepacia P. cepacia P. cepacia P. fluorescens P. fluorescens P. fluorescens
Methanol Methanol Methanol Methanol Ethanol Methanol Methanol Methanol
Soybean Soybean Soybean Palm kernel Palm kernel Soybean Soybean Sunflower
35 35 35 40 40 35 35 45
90 90 90 8 8 90 90 5
P. fluorescens
Ethanol
Sunflower
45
5
R. miehei
Methanol
Sunflower
40
48
R. oryzae T. lanuginosus
Methanol Methanol
Soybean Sunflower
– 40
Immobilized lipases C. antarctica Methanol
Temp. Time Acyl acceptor: [°C] [h] oil ratio
Solvent
Yield Reusability [wt.%]
Reference
>80 >80 100 15 72 >80 >80 79
n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Kaieda et al. 2001 Kaieda et al. 2001 Kaieda et al. 2001 Abigor et al. 2000 Abigor et al. 2000 Kaieda et al. 2001 Kaieda et al. 2001 Mittelbach 1990
99
n.d.
Mittelbach 1990
6:1 (1 portion)
– – – – – – – Petroleum ether Petroleum ether –
95.5
n.d.
70 48
3:1 (2 portions) 6:1 (1 portion)
– –
90.5 92.3
n.d. n.d.
Oliveira and Rosa 2006 Kaieda et al. 1999 Oliveira and Rosa 2006
30
48
3:1 (3 portions)
–
97.4
104 days
Shimada et al. 1999
30
34
3:1 (2 portions)
–
96.8
105 days
Watanabe et al. 2000
45
5
53
n.d.
Mittelbach 1990
79
n.d.
Mittelbach 1990
3:1 (2 portions) 3:1 (2 portions) 3:1 (3 portions) 4:1 4:1 3:1 (2 portions) 3:1 (2 portions) 10,8:1 10,8:1
C. antarctica
Methanol
C. antarctica
Methanol
Soybean+ rapeseed Soybean+ rapeseed Soybean
C. antarctica
Ethanol
Soybean
45
C. C. C. C.
Methanol Methanol Methanol Ethanol
Soybean Soybean Soybean Soybean
30 30 40 40
C. antarctica
Ethanol
Soybean
65
6
C. antarctica
Methyl acetate Soybean
40
14
12:1 (3 portions)
C. antarctica M. miehei M. miehei
Ethyl acetate Methanol Methanol
Sunflower Soybean Soybean
50 45 45
12 5 5
92.7 75.4 25
M. miehei
Ethanol
Soybean
45
5
82
n.d.
Mittelbach 1990
M. miehei
Ethanol
Soybean
35
8
11:1 – 3:1 (3 portions) – 10.8:1 (1 portion) Petroleum ether 10.8:1 (1 portion) Petroleum ether 3:1 (1 portion) n-Hexane
Rodrigues et al. 2008 Rodrigues et al. 2008 Wang et al. 2006 Oliveira and Oliveira 2001 n.d. Habulin and Knez 2001 >100 cycles Du et al. 2004; Xu et al. 2003 n.d. Kim et al. 2007 n.d. Nelson et al. 1996 n.d. Mittelbach 1990
95.6
n.d.
P. cepacia R. delemar R. miehei
Methanol Methanol Methanol
Soybean Soybean Soybean
45 45 30
8 5 6
3:1 (3 portions) 3:1 (3 portions) 7.5:1 (1 portion)
– – n-Hexane
14.5 0.8 ∼7
n.d. n.d. 7 cycles
T. lanuginosus
Methanol
Sunflower
30
6
7.5:1
–
16
n.d.
T. lanuginosus
Ethanol
Sunflower
30
6
7.5:1
–
35
n.d.
Rapeseed
35
12
4:1 (1 portion)
t-Butanol
95
>100 days
De Oliveira et al. 2005 Nelson et al. 1996 Nelson et al. 1996 Rodrigues et al. 2008 Rodrigues et al. 2008 Rodrigues et al. 2008 Li et al. 2006
Rapeseed
35
4
2:1 (1 portion)
t-Butanol
33.5
>200 cycles Du et al. 2007
antarctica antarctica antarctica antarctica
T. lanuginosus+ Methanol C. antarctica T. lanuginosus Methanol
10.8:1 (1 portion) petroleum ether 5 10.8:1 (1 portion) petroleum ether 6 7.5:1 (1 portion) n–Hexan 6 5:1 (1 portion) n-Hexan 25 3.9:1 (1 portion) t-Butanol n.d. 10:1 SCCO2 6:1
comp. propan -
∼35 ∼45 94 63.2 100 92
7 cycles 7 cycles n.d. n.d.
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Table 2 (continued) Origin of lipase
Acyl acceptor
Oil source
Temp. Time Acyl acceptor: [°C] [h] oil ratio
Solvent
T. lanuginosus T. lanuginosus
Methanol Methanol
Rapeseed Rapeseed
35 35
4 4
2:1 (1 portion) 2:1 (1 portion)
T. lanuginosus
Methanol
Soybean
30
6
7.5:1 (1 portion)
Yield Reusability [wt.%]
n-Hexan Petroleum ether n-Hexane
37.5 37.4 ∼18
Reference
∼10 cycles ∼10 cycles
Du et al. 2007 Du et al. 2007
7 cycles
Rodrigues et al. 2008
C. Candida, comp. compressed, M. Mucor, n.d. no data, P. Pseudomonas, R. Rhizopus, SCCO2 supercritical CO2, T. Thermomyces, Temp. temperature
dithio wax esters was reported (Kalscheuer et al. 2003; Uthoff et al. 2005). Figure 5 shows the WS/DGAT-catalyzed reaction responsible for FAAE synthesis. An acyl-CoA thio ester is esterified with a short chain alcohol, forming FAAE and Coenzyme A. To simplify the process, the three required genes atfA, pdc, and adhB were combined on the plasmid pMicrodiesel, which is a derivative of the high-copy-number vector pBluescript SK−, including two lacZ promoters, one upstream of atfA and the other upstream of the pdcadhB operon (Kalscheuer et al. 2006). In these first experiments, the final FAEE concentration reached 1.28 g L−1, and the productivity was 0.0178 g·(L h)−1,
respectively. Hence, 62.7% of the externally added oleic acid was converted to ethyl esters yielding a cellular FAEE content of 26% (Kalscheuer et al. 2006). Recently, the recombinant pMicrodiesel-containing strain of E. coli was cultivated in a 20-L fed-batch bioreactor yielding a conversion rate of 75%, a FAEE concentration of 12.6 g·L−1, a FAEE content of the cells of 23.4% of the cellular dry mass, and a productivity of 0.19 g·(L·h)−1 (Elbahloul and Steinbüchel, unpublished). This clearly shows that the approach is feasible in E. coli although the yield has to be further optimized. The company LS9 Inc. (South San Francisco, USA) developed further approaches to synthesize FAAE de novo by introducing several genes encoding for enzymes like
Table 3 Transesterifications of TAGs to FAAE by whole-cell catalysts Organism
Localisation of lipase
Oil
Temp. [°C]
Time [h]
Alcohol/ oil ratio
Solvent
R. oryzae R. oryzae R. oryzae
Intracellular Intracellular Intracellular
n.d. 35 35
n.d. 24 24
3:1 3:1 3:1
t-Butanol t-Butanol t-Butanol
72 60 70
Li et al. 2007a Li et al. 2007b Li et al. 2007b
R. oryzae R. oryzae
Intracellular Intracellular
35 35
24 24
3:1 4:1
t-Butanol t-Butanol
60 70
Li et al. 2007b Li et al. 2007b
R. R. R. R.
Intracellular Intracellular Intracellular Intracellular
Soybean Rapeseed (raw) Rapeseed (acidulated) Rapeseed (refined) Rapeseed (refined)+ FFA Soybean Soybean Soybean Rapeseed (raw)
32 35 30 25
72 72 96 72
3:1 3:1 3:1 3:1
– –
80–90 70–83 94 90
Ban et al. 2001 Ban et al. 2002 Hama et al. 2008 Jin et al. 2008
Intracellular Intracellular
Waste Soybean
25 37
72 165
3:1 3:1
– –
80 71
Cell surface
Soybean
37
12
3:1
–
78.6
Intracellular Intracellular Intracellular Intracellular
Soybean Olive Rapeseed Soybean
30 30 15 15
72 20 12 12
3:1 5:1 5:1 5:1
– – – –
86 100 97.7 95
Jin et al. 2009 Matsumoto et al. 2001 Matsumoto et al. 2002 He et al. 2008 Gao et al. 2009 Gao et al. 2009 Gao et al. 2009
oryzae oryzae oryzae oryzae
R. oryzae S. cerevisiae (+ R. oryzae lipase) S. cerevisiae (+ R. oryzae lipase) R. chinesis E. coli E. coli E. coli
E. Escherichia, FFA free fatty acids, Temp. temperature, n.d. no data, R. Rhizopus, S. Saccharomyces
Yield [wt.%]
Reference
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Fig. 5 Enzymatic esterification of fatty acid acyl-Coenzyme A by the acyltransferase WS/DGAT. CoA Coenzyme A, R1 fatty acid residue, R2 alcohol moiety
thioesterase, wax synthase, alcohol acyltransferase, alcohol dehydrogenase, and different kinds of fatty alcohol forming acyl-CoA reductases in microbial host cells (Del Cardayre et al. 2009).
Other in vivo synthesis routes of FAAE occurring in natural organisms, their applications and relevance Several microorganisms, plants, insects, and mammalian species possess the ability to synthesize FAEE, e.g., many yeasts and filamentous fungi such as Rhizopus arrhizus and Neurospora sp. For some beetles and bees like Trogoderma garnarium or Bombus terrestris, FAEE serve as pheromones. Furthermore, they are synthesized in mammals in consequence to ethanol consumption (Best and Laposata 2003; Park et al. 2009). In addition, FAEE are used as aroma additives in food, beverages, or cosmetics (Liu et al. 2004). Besides, they serve as dietary supplements to enable an increased uptake of constitutional polyunsaturated fatty acids (Armenta et al. 2007). The examples described below for FAEE formation may not be directly relevant for the biotechnological production of FAEE, but they point to enzymes that could be involved in respective pathways of microorganisms engineered for FAEE production. FAEE biosynthesis in S. cerevisiae During alcoholic fermentation, S. cerevisiae and other yeasts-synthesize ethyl esters from ethanol and acetate and, to a lower extent, also ethyl esters from medium carbon chain length fatty acids (MCFA). These acetate and ethyl esters possess different fruity aromas and affect therefore greatly the flavor of fermented and distilled alcoholic beverages like beer, wine, or scotch whiskey (Goss et al. 1999; Saerens et al. 2008). In general, biosynthesis of these esters is often catalyzed by alcohol acyltransferases (AATases); with AATases of different species differing in cell localization and substrate specificity. For example, the Neurospora AATase exhibits high activities towards coenzyme A derivatives of MCFA like hexadecanoyl-CoA and longer chains (Park et al. 2009). In S. cerevisiae, acetyl-CoA:isoamyl alcohol acetyltransferase (ATF1) catalyzes the formation of isoamyl acetate and ethyl acetate and is mostly responsible for
Appl Microbiol Biotechnol (2010) 85:1713–1733
acetate ester formation. The contribution of ATF2, which shows an amino acid similarity of 37%, is yet uncertain (Mason and Dufour 2000). Since ethyl acetate is still detectable in atf1 atf2 double mutants, there are probably other enzymatic routes to generate this ester (Verstrepen et al. 2003). Furthermore, numerous orthologs in other Saccharomyces species, as well as in Torulopsis castelii, Candida glabrata, Kluyveromyces waltii, and K. lactis have been found (Van Laere et al. 2008). The regulation of AATases, and thus the amount of aromatic isoamyl acetate and ethyl acetate, is very complex as the enzymes can be repressed by oxygen or exogenous (poly)unsaturated fatty acids (Fujii et al. 1997). Must of grapes grown in cold regions contains an increased level of the polyunsaturated linoleic and linolenic acid which hinders the FAEE formation during fermentation, and thus affects the aroma profile of the resulting wine (Yunoki et al. 2005, 2007). Acetate esters of aromatic relevance are, for example, isoamyl acetate, which has banana aroma or phenylethyl acetate, which smells like roses and honey. Besides AATases other enzymes of S. cerevisiae like Adh are capable of catalyzing ester formation, which oxidize hemiacetal hydroxyl groups yielding esters like methyl or ethyl acetate. This side reaction corresponds to the oxidation of secondary alcohols and includes reduction of NAD(P)+ (Park et al. 2009). In C. utilis, Adh is presumably even of greater importance than AATase activity for ethyl acetate formation (Kusano et al. 1999). MCFA ethyl esters are formed by the two acyl-CoA: ethanol O-acyltransferases Eeb1 and Eht1. Eht1 prefers short-carbon-chain length substrates (like butyryl-CoA) whereas Eeb1 prefers substrates with longer carbon-chain length (like hexanoyl- or octanoyl-CoA) and mostly contributes to biosynthesis of MCFA ethyl esters. Orthologous enzymes have been identified in different fungi, and the existence of additional enzymes with the ability to synthesize ethyl esters is expected because these substances are still detectable in eeb1 eht1 double mutants of S. cerevisiae (Saerens et al. 2006). A possible physiological function of these enzymes might be in the cellular lipid metabolism and the detoxification of MCFA. The latter can be prematurely released during fatty acid biosynthesis and are toxic for the yeast cell (Saerens et al. 2006; Stratford and Anslow 1996). Ethyl esters of aromatic relevance are ethyl hexanoate and ethyl octanoate with a fruity apple aroma, as well as ethyl decanoate with a flowery aroma (Fig. 6). The extent of ethyl ester synthesis during fermentation depends on the particular yeast strain, the composition of the medium, and the fermentation conditions like temperature, rate of aeration, etc. (Saerens et al. 2008). Furthermore, it was found that long-chain fatty acids (C14 and higher), that are mainly derived from grape must, accumu-
Appl Microbiol Biotechnol (2010) 85:1713–1733
Fig. 6 Chemical structures of aroma-relevant FAEE
late in yeast cells as ethyl esters (Yunoki et al. 2007). Since the fruity- and apple-like aromas of many FAEE are desirable for wine production, the mechanisms of ethyl ester synthesis in yeasts is currently investigated to increase their production during fermentation (Saerens et al. 2008). In addition to modification of culture conditions, genetic modifications of yeast strains, like an overexpression of atf1 or eht1, leads to enhanced levels of ethyl esters (Lilly et al. 2006). FAEE biosynthesis in mammals (after ethanol ingestion) Mammals possess oxidative and non-oxidative metabolic pathways to degrade ethanol. Oxidative degradation to acetaldehyde or acetate mainly occurs in the liver (Best and Laposata 2003; Riveros-Rosas et al. 1997). However, in many other organs like pancreas, heart, or brain, nonoxidative pathways are of greater relevance, which result in the ethylation of endogenous fatty acids. The esterification of free fatty acids and ethanol is catalyzed by cytosolic FAEE synthases whereas CoA-activated fatty acids are ethylated via the microsomal, membrane bound acyl-CoA: ethanol O-acyltransferase (AEAT; Best and Laposata 2003; Treloar et al. 1996). In most human tissues, AEAT activity is much higher than FAEE synthase activity. For example, AEAT activity in human liver homogenates was 545 nmol/ min/g tissue, whereas FAEE synthase activity was only 60 nmol/min/g tissue (Diczfalusy et al. 2001). So far, particular gene products that are responsible for AEAT activity were not characterized in detail. It remains uncertain, whether the detected enzyme activities for FAEE synthesis are exhibited by enzymes whose natural function is FAEE synthesis or by enzymes with another primary function, e.g., carboxylesterase or lipase, lipoprotein lipase, or cholesterol esterase (Best and Laposata 2003; Salem and Laposata 2005). However, in many studies, esterification enzymes were responsible for the measured FAEE synthase activities in various tissues (Salem and Laposata 2005). For example, it was demonstrated that purified carboxylesterase
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promotes FAEE synthesis from TAG and ethanol (Tsujita and Okuda 1994). Furthermore, carboxylesterase is structurally and functionally similar to FAEE synthase (Kaphalia et al. 1997). These non-FAEE specific enzymes hydrolyze fatty acids from TAGs or phospholipids and esterify them to FAEE when ethanol is present (Best and Laposata 2003). It is assumed that FAEE are critical mediators of ethanolinduced cytotoxicity and damage of pancreas, liver, heart, or brain as these organs do not show an oxidative degradation pathways for ethanol but a comparatively high FAEE synthase activity after ethanol consumption. Chronic ethanol uptake leads to an accumulation of FAEE in the adipose tissue because of a similar hydrophobicity of FAEE and TAGs (Laposata and Lange 1986). To date, many studies have demonstrated the toxicity of FAEE in vitro as well as in vivo (Alhomsi et al. 2008; Gorski et al. 1996; Gubitosi-Klug and Gross 1996; Haber et al. 1993; Hungund et al. 1988; Szczepiorkowski et al. 1995; Werner et al. 1997). Orally ingested FAEE are not toxic as they are hydrolyzed rapidly in the stomach and duodenum by unspecific hydrolases (Saghir et al. 1997). FAEE are discussed as suitable markers for ethanol intake because there is a high correlation between the amounts of ethanol and FAEE in blood. But unlike ethanol, FAEE are still detectable up to 24 h after ethanol intake (Salem and Laposata 2005). In case of autopsy, the FAEE concentration in adipose or liver tissue can give important information about an influence of alcohol to the time of death, primarily if blood samples are not available anymore (Refaai et al. 2002).
Outlook and perspectives Current processes of biodiesel production are not environmentally compatible and do not provide an appropriate net energy gain since they require expensive feedstocks, several energy-consuming process steps, and high temperatures. Furthermore, they rely on petrochemically derived methanol, although ethanol is more sustainable and the resulting esters have better fuel properties. Microalgaederived oil seems to be superior to vegetable oils as it could solve the problem of limited agricultural land and the low yield per hectare of crops. Nevertheless, microalgae-derived TAGs still require energy consuming extraction, purification, and transesterification process. The entire substitution of fossil fuels would only be feasible, if abundant, low cost biomass were used for biodiesel synthesis (Kalscheuer et al. 2007). The crucial step is the release of monosaccharides and other microbially utilizable building blocks from lignocellulose since the biomass pretreatment is time- and energy-consuming (Himmel et al. 2007). Therefore, many approaches try to
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confer the capability of biomass degradation to biofuel production strains or to facilitate cellulolytic organisms to synthesize biofuels by metabolic engineering (Fischer et al. 2008). Moreover, the composition of microbial synthesized FAAE (e.g., the chain length and degree of saturation) could easily be tailored by genetic engineering of wellstudied microorganisms, whereas the composition of plantderived biodiesel cannot be that easily altered (Rude and Schirmer 2009). In consequence, an intracellular generation of microdiesel from simple carbon sources, as it was established by Kalscheuer et al. (2006) is an important initial step. Since E. coli is unable to metabolize lignocellulose, and since its de novo fatty acid biosynthesis hardly provides an excess of fatty acids, it is not an ideal host for microdiesel production. A recently constructed broad host range plasmid, which contains all relevant genes (atfA, adhB, and pdc) for microdiesel production, can establish in vivo FAEE production in other biotechnologically relevant organisms like Pseudomonas putida or Ralstonia eutropha (Röttig and Wenning, unpublished). A further promising research objective may be the transfer of this broad host range plasmid to other microorganisms, which degrade and metabolize lignocellulose to synthesize ethanol and large quantities of fatty acids or TAG. Lu and colleagues showed for E. coli that an overproduction of FFA can be achieved by metabolic engineering. This approach could also be suitable for other putative production organisms (Lu et al. 2008). Furthermore, other alcohols like (iso-)propanol or (iso-)butanol, which are for example obtained by fermentation, may be esterified in vivo resulting in FAAE with enhanced properties. This would constitute a more adequate diesel substitute with better cold flow properties. In a final step, the establishment of a secretion module for intracellularly formed FAAE is desirable. Secretion of TAGs and other lipids has been observed in species of the genus Alcanivorax (Bredemeier et al. 2003; Yakimov et al. 1998). This would allow a further reduction of costs and a continuous production of microdiesel (Kalscheuer et al. 2007).
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