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Biodiesel Production and Technologies
Biodiesel Production and Technologies Jingyong Yan and Yunjun Yan, Huazhong University of Science and Technology, Wuhan, PR China Ó 2017 Elsevier Inc. All rights reserved.
Introduction As an alternative to fossil fuels, green and clean biofuels must be technically feasible, economically competitive, and environmentally acceptable. One of them, biodiesel, an alternative diesel fuel, is made from renewable sources of oils and fats. It is biodegradable and nontoxic, with a low emission profile, so it is environmentally beneficial. Biodiesel is a mixture of mono-alkyl esters produced from vegetable oils, animal fats, forestry oils, waste cooking oils, algae oils, etc. In recent decades, biodiesel has been drawing wide attention for its ability to replace fossil fuels, which are likely to be exhausted in the near future. Additionally, the environmental issues caused by emission of gases from combustion of fossil fuels have also encouraged development of biodiesel, which is considered to be a carbon neutral fuel, because its carbon content is originally fixed from the atmosphere. In general, to develop biodiesel from biological resources is of great significance to the development of mankind. One hundred years ago, Rudolf Diesel tested vegetable oil as fuel. With the advent of cheap petroleum, appropriate crude oil fractions were refined to serve as fuel, and diesel fuels and diesel engines evolved together. In the 1930s to 1940s, vegetable oils were used as diesel fuels usually only in emergency situations. Recently, because of increases in crude oil prices, limited resources of fossil oils, and environmental concerns, there has been a renewed focus on plant oils and animal fats to make biodiesel fuels. Continuing and increasing use of petroleum will intensify local air pollution and magnify the global warming problems caused by CO2. In a particular case, such as the emission of pollutants in the closed environments of underground mines, biodiesel fuel has the potential to reduce the level of pollutants and the level of potential or probable carcinogens (Krawczyk, 1996). Fats and oils are primarily water-insoluble, hydrophobic substances in the plant and animal kingdoms that are made up of one mole of glycerol and three moles of fatty acids (commonly referred to as triglycerides, TGs). Fatty acids vary in carbon chain length and in the number of unsaturated bonds (double bonds). For example, in beef tallow, the saturated fatty acid component accounts for almost 50% of the total fatty acids, while in soybean oil, the unsaturated fatty acid component accounts for almost 85%. However, the higher stearic and palmitic acid contents give beef tallow the unique properties of a higher melting point and higher viscosity compared to soybean oil. Table 1 shows typical fatty acid compositions of common oil sources. As is known, the kinematic viscosity of oils and fats is much higher than that of diesel, owing to their higher molecular weight and complex structure. In order to reduce viscosity of oils and fats, alcohol is usually employed to replace glycerol via transesterification reaction. Therefore, biodiesel can be synthesized by chemically combining any natural oils or fats with an alcohol such as methanol or ethanol by the catalysis of an alkali/acid or a lipase (see Fig. 1). Methanol and ethanol are the most commonly used alcohols in the commercial production of biodiesel. Though biodiesels are esters of natural oils, different oils retain similar chemical and physical properties when they are transesterified. So far, abundant research has proven that biodiesel made from plant oils and animal fats can be effectively used in diesel engines without any modification. Moreover, the energy density of biodiesel is very close to fossil diesel. Similar combustion properties between biodiesel and petroleum-derived diesel have made the former one of the most promising renewable and sustainable fuels for the automobile.
Feedstock of Biodiesel Vegetable oils and animal fats are extracted, or pressed, to get crude oil or fat. These usually contain free fatty acids, phospholipids, sterols, water, odorants, and other impurities. Even the refined oils and fats also retain small amounts of free fatty acids and water. Table 1
Typical fatty acid compositions of common oil sources
Oils
C16:0
Corn Rapeseed Soybean Peanut Sesame Rice bran Wheat grain Sunflower Groundnut Crambe Linseed Safflower Cottonseed
11.7 3.5 11.4 11.4 13.0 11.7–16.5 20.6 7.1 8.5 2.1 5.0 7.3 28.3
C16:1
C18:0
C18:1
C18:2
C18:3
C20:0
25.2 64.4 20.8 48.3 53.0 39.2–43.7 16.6 25.5 51.6 18.9 20.0 13.5 13.3
60.6 22.3 53.8 32.0 30.0 26.4–35.1 56.0 62.4 26.0 9.0 18.0 77.0 57.5
0.5 8.2 9.3 0.9
0.2
1.0
1.9 0.9 4.4 2.4 4.0 1.7–20.5 1.1 4.7 6.0 0.7 2.0 1.9 0.9
0.1
Encyclopedia of Sustainable Technologies, Volume 3
0.3 1.3
C20:1
C22:0
C22:1
2.5
n9
C24:0
Others
1.2
0.4–0.6
0.4–0.9
2.9
1.8 0.3
6.9 55.0
2.1
http://dx.doi.org/10.1016/B978-0-12-409548-9.10110-1
0.8
58.5
1.1 0.2
261
262
Fig. 1
Biodiesel Production and Technologies
Formula of biodiesel production with short chain alcohols as acyl acceptors.
The free fatty acid and water contents have significant effects on the transesterification reaction of glycerides with alcohols ether using alkaline or acid as the catalyst. They also make the procedure of separating fatty acid esters and glycerol complex and very difficult. Considerable research has been done on using plant oils, especially vegetable oils, including palm oil, soybean oil, sunflower oil, coconut oil, rapeseed oil, tung oil, etc., as diesel fuel. Animal fats, although mentioned frequently, have not been studied to the same extent as vegetable oils. Some methods applied to vegetable oils are not applicable to animal fats because of physiochemical property differences. Recently, oils from algae, bacteria, and fungi have also been investigated. Microalgae, in particular, can grow very fast, resulting in a very short life cycle, but with very high oil content. They are being regarded as an ideal resource as biodiesel feedstock. Oils from microalgae have been examined as a source of methyl ester diesel fuel (Nagel and Lemke, 1990). In addition, terpenes and latexes are occasionally studied as diesel fuels (Calvin, 1985). An important reason to screen feedstock is that the biodiesel production cost is mainly dependent on feedstock price. Despite the widespread use of vegetable oils such as soybean oils and rapeseed oils for production of biodiesel, increasing competition between the production of biofuels and food has limited further application on an industrial scale, especially in developing countries. In this context, use of cost-effective oil resources replacing edible oils for biodiesel production is more economically viable. Waste cooking oils - abundant and cheap wastes from urban catering - have been widely used for synthesis of biodiesel. However, waste cooking oils generally contain large amount of free fatty acids. Therefore, during conversion of waste cooking oils into biodiesel, transesterification of triacylglycerols and esterification of free fatty acids are both performed. Alkali-catalyzed biodiesel production from waste cooking oils is not technically feasible without neutralization in the pretreatment step, because the saponification reaction between alkali catalyst and free fatty acid would limit a further conversion process. Lipase is feasible to convert these oils containing high free fatty acid into biodiesel because of its ability to simultaneously catalyze transesterification and esterification reactions. Other nonedible oils suitable for biodiesel production are summarized in Table 2. Microorganism derived oils (also called single cell oils), generated by oleaginous yeast, fungi, bacteria, and microalgae, are regarded as emerging feedstock for biodiesel production (Table 3). These oleaginous microorganisms generally accumulate lipids in the main form of triacylglycerols accounting to more than 20% of its dry cell biomass. Yarrowia lipolytica is a well known superior oleaginous yeast, which can generate than 30% of its dry cell weight in oil. Moreover, Y. lipolytica yeast is generally recognized as safe, capable of growing well on low cost substrates such as residues from agriculture and industry. Even better, the lipid biosynthetic pathway of the yeast can be genetically modified for enhancing lipid production by metabolic engineering and reconstruction. Thus, Y. lipolytica yeast has been used as a chassis for constructing cell factories for oil production.
Technologies for Biodiesel Production There are three major conversion technologies for biodiesel production: acid/base catalysis, enzyme catalysis, and supercritical fluid (SCF) technology. Acid/base catalysis has high requirements for water content and acid value of raw materials, and certain pollutants are produced. Enzyme catalysis takes a relatively long time, and the cost of the enzyme is rather high. The SCF method has recently emerged as a method of biodiesel production, but the technology is still in its infancy, and the process is expensive and complex. Therefore, development of green catalysts and ideal technologies will take time. However, technologies for biodiesel have been in development for a long time.
Direct Use and Blending The properties of heat content (80% of diesel fuel), availability, and renewability, make vegetable oils a potential alternative for diesel fuel. It is not recommended to use 100% oil for diesel fuel, because of its high viscosity. People found that this problem can be minimized by blending vegetable oils with pure diesel. Using a vegetable oils/diesel fuel blend is the simplest means to reduce the viscosity of vegetable oil, with no need for any chemical procedure. So far, blends of conventional diesel fuel with sunflower oil, soybean oil, cottonseed oil, rapeseed oil, Jatropha curcas oil, and Pongamia pinnata oil have been reported. Blending waste cooking oils and diesel with a suitable ratio has also been applied. A mixture of 20% vegetable oil and 80% diesel fuel was confirmed feasible, and some experiments even ran up to 50/50. However, obvious disadvantages, such as higher viscosity, lower volatility, and the reactivity of unsaturated hydrocarbon chains, limit widespread application. Thus, this blend was considered not suitable for long-term use in a direct injection engine.
Biodiesel Production and Technologies
Table 2
263
Biodiesel production from feedstock of nonedible oils
Nonedible oils
Genus/species
Distribution
Balanos oil Bladderpod oil
Balanites aegyptiaca Physaria
Castor oil Chaulmoogra oil Copaiba Crambe oil Croton oil Cuphea oil Honesty oil Honge oil Illipe butter Jatropha oil Jojoba oil
Castor beans Hydnocarpus wightiana Copaifera Crambe abyssinica Croton tiglium Cuphea Lunaria annua Millettia pinnata Shorea stenoptera Euphorbiaceae Simmondsia chinensis
Mahua Mango oil Milk bush Nahor oil Neem oil Petroleum nut oil Rubber seed oil Sea buckthorn oil Stillingia oil Tall oil Tamanu Tonka bean oil Tung oil Vernonia oil
Madhuca indica Mangifera indica Euphorbia tirucalli Mesua ferrea L. Azadirachta indica Pittosporum resiniferum Rubber tree Hippophae rhamnoides Sapium sebiferum wood pulp manufacture Calophyllum inophyllum Dipteryx odorata Vernicia fordii Vernonia galamensis
Egypt Southwestern United States, eastward to Kansas and southward into northern Mexico Mediterranean Basin, Eastern Africa, and India Indonesia, Malaysia, and the Philippines Tropical Africa and North and South American Europe, southwest and central Asia, and eastern Africa India and the Malay Archipelago America South east Europe and western Asia Asia Southeast Asia, Borneo, and Sumatra India Southern Arizona, Southern California, and northwestern Mexico India India Africa Indian origin Indian subcontinent and other areas in the tropics Philippines Cambodia and Nigeria Europe and Asia China India and Nigeria Southeast Asia Northern South America China East Africa
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and, Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
Microemulsification of Oils Microemulsification is a method for forming microemulsions, which are transparent, thermodynamically stable colloidal dispersions. To solve the problem of the high viscosity of vegetable oils, microemulsions with immiscible liquids, such as methanol, ethanol, and ionic or nonionic amphiphiles have been investigated. It has been proven that short-term performances of both ionic and nonionic microemulsions of aqueous ethanol in soybean oil are comparable to that of No. 2 diesel fuel (Goering, et al., 1982). Ziejewski et al. (1984) conducted a microemulsion with 53% (v/v) alkali-refined and winterized sunflower oil, 13.3% (v/v) 190proof ethanol, and 33.4% (v/v) 1-butanol. This microemulsion possessed a viscosity of 6.31 cSt at 313 K, a cetane number of 25, and an ash content less than 0.01%. Lower viscosities and better spray patterns were observed with an increase of 1-butanol.
Thermal Cracking (Pyrolysis) Producing biofuels from vegetable oils by thermal cracking (pyrolysis) is a promising alternative. The purpose of pyrolysis is to optimize high-value fuel products from biomass through thermal means or coupling with catalysts. The potential materials can be any type of biomass, such as vegetable oils, animal fats, wood, biowaste, etc. Many studies have reported the pyrolysis of TGs to obtain products suitable for diesel engines. Thermal pyrolysis of TGs produces many compounds, including alkanes, alkenes, alkadienes, aromatics, and carboxylic acids. Different types of vegetable oils lead to significant differences in the composition of the pyrolyzed oil. The mechanism for the thermal decomposition of TGs is complex, owing to the structures and multiplicity of possible reactions of the mixed TGs. In general, pyrolysis of these structures occurs via either a free-radical or carbonium ion mechanism. Formation of aromatics is supposed by a Diels-Alder addition of ethylene to a conjugated diene formed in the pyrolysis reaction. The carboxylic acids formed during the pyrolysis of vegetable oils probably result from cleavage of the glyceride moiety. Until recently, several vegetable oils, such as palm oil, canola oil, and soybean oil, have been employed in the process involving conversion of the oils into biofuels suitable for gasoline engines using acid catalysts (transition metal catalysts which yield biofuels enriched in diesel fractiondover 50% by weight, and zeolites and mesoporous materials that give biofuels with higher gasoline fractionsdover 40%, with higher aromatic content) (Luque, et al., 2008). The reaction is usually conducted at moderate to high temperatures
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Table 3
Oil content of microorganisms
Microorganisms Microalgae Botryococcus braunii Chlorella sorokiana C. emersonii C. vulgaris Crypthecodium cohnii Cylindrotheca sp. Dunaliella salina Isochrysis galbana Monodus subterraneus Nannochloropsis oculata Neochloris oleoabundans Nitzschia sp. N. laevis Phaeodactylum tricornutum Porphyridium cruentum Parietochloris incise Scenedesmus dimorphus S. obliquus Schizochytrium sp. Spirulina platensis Bacterium Arthrobacter sp. Acinetobacter calcoaceticus Bacillus alcalophilus Rhodococcus opacus Yeast Candida curvata Candida sp. 107 C. valida C. utilis C. curvatus Cryptococcus albidus Lipomyces starkeyi Rhodotorula glutinis R. graminis Rhizopus arrhizus Rhodosporidium toruloides Trichosporon pullulans Yarrowia Lipolytica Fungi Aspergillus oryzae Cunninghamella japonica C. bainieri Humicola lanuginose Mucor circinelloides M. rouxii Mortierella isabellina M. vinacea M. ramanniana
Oil content (% dry cell weight)
25–75 22 29.63 56.6 56 16–37 14–20 14.5 39.3 22–44 35–65 45–47 69.1 6–7 19.3 62 16–40 35–55 50–77 5–17 >40 27–38 18–40 24–25 58 42 26–37 64 72 65 63 72 36 57 48–67.5 65 36 57 50 38 75 23 32 86 66 54.2
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
(573–773 K) using different oils/catalyst ratios, depending on the oil and the catalyst. Catalytic cracking not only increases the yield of gasoline by breaking large molecules into smaller ones, but also improves the quality of the gasoline: this process involves carbocations and yields alkanes and alkenes with the highly branched structures desirable in gasoline. The main components are alkanes and alkenes, which account for 60% of the total weight. Carboxylic acids account for another 9.6%–16.1% (Ma and Hanna, 1999a, 1999b). However, the heterogeneous products and high temperature are the main drawbacks for cracking oils into diesel target product. It is also difficult to control selectivity of cracking products.
Biodiesel Production and Technologies
265
Transesterification (Alcoholysis) Transesterification reaction is the use of a kind of oil and an alcohol as substrates to prepare esters and glycerol. The reversible reaction process is also called alcoholysis. To drive the equilibrium to the product direction, excess alcohol is often loaded. Various oilrich feedstocks, including vegetable oils, waste oils, and microalge or microorganism derived oils, and methanol or ethanol are frequently used for biodiesel production. A catalyst responsible for improving the reaction rate and biodiesel yield can be alkalis, acids, or enzymes.
Transesterification catalyzed by chemical catalysts As for chemical-catalyzed biodiesel production via transesterification reaction, NaOH, KOH, carbonates and corresponding sodium and potassium alkoxides, such as sodium methoxide, are frequently served as alkali catalysts. Sulfuric acid, sulfonic acids, and hydrochloric acid are usual acid catalysts. Various chemical catalyses for biodiesel production on reactors are summarized in Table 4.
Acid-catalytic transesterification methods Sulfuric, phosphoric, hydrochloric, and organic sulfonic acids can be used to catalyze transesterification reaction to produce biodiesel. Currently, the more-used catalysts in biodiesel production are organic acids: for example, the derivates of toluenesulfonic acid and, more often, mineral acids such as sulfuric acid. Usually, the transesterification rate if using acid catalysts is much slower than that with alkali catalysis. But if high contents of water and FFAs are present in the vegetable oil, acid-catalyzed transesterification can be used. The acid-catalyzed reaction commonly needs high temperatures beyond 373 K and reaction times of 3–48 h. These catalysts give fairly high yields in the transesterification process. Acid-catalyzed reactions also require the use of high alcohol-to-oil molar ratios in order to obtain ideal product yields in the practical reaction. However, ester yields do not proportionally increase with molar ratio. Rachmaniah et al. (2004) investigated transesterification of the refined soybean oil (> 99% TGs) and low grade high FFA (60%) rice bran oil with methanol by using 10% hydrochloric acid as a catalyst (w/w) at a temperature of 343 K. In their study, all reactions were conducted with a methanol molar ratio of 20:1. The highest FAME content of 90% was obtained from transesterification of low grade high FFA rice bran oil with a reaction time of 6 h.
Alkali-catalytic transesterification methods Alkali-catalytic transesterification is catalyzed by alkaline metal alkoxides and hydroxides, as well as sodium or potassium carbonates. Sodium methoxide is the most widely used biodiesel catalyst. Alkaline catalysts have the following advantages: (a) short reaction time; (b) relatively low temperature; (c) only a small amount of catalyst used; and (d) little or no darkening of color of the oil. Nowadays, among commercialization technologies of biodiesel production, alkali-catalyzed transesterification is the main feasible route because of the above merits. However, for the oils containing significant amounts of FFAs, alkaline catalysts do not convert it to biodiesel, but rather into a lot of soap. These FFAs react with the alkaline catalyst to produce soaps that inhibit the separation of the biodiesel, glycerin, and wash water. In alkali-catalytic transesterification, the catalyst is first dissolved into alcohol by vigorous stirring, and then the catalyst/alcohol mixture is pumped into oil to react and stirred vigorously for 2 h at 340 K in ambient pressure. A successful transesterification reaction produces two liquid phases: ester and crude glycerol. Generally, the alkali-catalyzed transesterification process is conducted at low temperatures and pressures (333–338 K and 1.4–4.2 bar) with low catalyst concentrations (0.5–2 wt%), and a conversion rate of over 95% can be expected after 1 h reaction. Although the homogeneous-catalyzed biodiesel reaction is relatively fast and results in high conversions, it has some serious drawbacks: (a) the catalyst cannot be recovered; (b) it must be neutralized and separated from the methyl ester phase at the end of the reaction; and (c) it consequently generates a large volume of wastewater. The above problems have provided an impetus to search for new stable and more environmentally friendly solid catalysts. For searches of promising solid base catalysts, attempts were made with alkali earth oxides, such as CaO, SrO, and BaO. Alkali and alkali earth salts loaded on metal oxide such as KOH/ Al2O3, KF/MgO, Sr(NO)3/ZnO, Ca(NO)3/Al2O3, and calcined Mg–Al hydrotalcites. On the other hand, a variety of solid acids has already been tested, including carbon-based solid acid, WO3/ZrO2, SO4/ZrO2, SO4/TiO2, SO4/ZrO2-Al2O3, and Nafion. They can Table 4
Representative chemical catalysis for biodiesel production on reactors
Reactor type
Feedstock
Catalyst
FAME yield (%)
Membrane reactor Zigzag micro-channel reactor Oscillatory flow reactor Trickle bed reactor rotating packed bed Stirred-tank reactor Reactive distillation reactor
Canola oil, methanol Soybean oil, methanol Rapeseed oil, methanol Rapeseed oil, methanol soybean oil methanol Palm oil, methanol Canola oil, methanol
NaOH, H2SO4 NaOH NaOH Alkaline KOH KOH KOH
96, 64 81.5–99.5 99 95 97.3 58.5–97.3 94.4
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
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obtain catalytic efficiency similar to those of homogeneous catalysts. What’s more, they endow the catalyst recovery property. Nevertheless, the chemical route for biodiesel production generally requires higher energy input than the enzymatic route.
Transesterification catalyzed by lipases Although the alkali-catalytic transesterification process gives high conversion of TGs to biodiesel in short reaction times, it suffers from several shortcomings: (a) it is energy intensive; (b) the recovery of glycerol is difficult; (c) the alkaline catalyst has to be removed from the product; (d) alkaline wastewater requires treatment; and (e) FFAs and water interfere with the reaction. Enzymatic catalysts like lipases are able to effectively catalyze the transesterification of TGs in either aqueous or nonaqueous systems. Thus, they can overcome the above-mentioned problems. Particularly, the byproduct, glycerol, can be easily recovered with a simple separation process. However, enzymatic catalysts are often more expensive than chemical catalysts, so recycling and reusing strategies are often applied for commercial viability. Though the high cost of enzyme production is a major obstacle to the commercialization of enzyme-catalyzed processes, recent advances in enzyme technology, such as use of solvent-tolerant lipases (especially immobilized lipases), making catalyst reutilization possible, has developed cost-effective systems. It can be seen that enzymatic biodiesel production by lipase catalyzed transesterification is a clean and green route. An enzymatic route with mild reaction conditions and a stoichiometrical amount of alcohol dosage show obvious advantages over chemcal routes. Especially, simultaneous esterification of free fatty acid and transesterification with triacylglycerol for biodiesel production can be achieved by lipase mediated biotransformation. Various lipases from bacteria such as Burkholderia cepacia, yeast such as Candida antarctica, and filamentous fungus such as Thermomyces lanuginosus have often been used for this process (Table 5). Several lipases for biodiesel production are commercially available (Table 6). Biocatalyst cost is one of the main factors affecting the overall cost of biodoesel production. Overexpression of desirable lipases in genetically tractable hosts such as E. coli, Bacillus subtilis, and Pichia pastoris is an effective strategy to lower the biocatalyst cost. Additionally, immobilization of lipases can further reduce the biocatalyst cost due to improved tolerance to solvents and recyclability. Various materials and technologies have been developed for immobilization of lipases, and great progress has been made. Alternatively, lipases in the forms of whole cells, including intracellular type, surface displayed type, and extracellular type, have been widely used to catalyze biodiesel production (Table 7, Fig. 2). Advantage and drawback comparison of different engineered microbial whole cells used for biodiesel production is summarized in Table 8.
Transesterification via solvent engineering Although biodiesel production in solvent-free systems is extensively reported, lipase inactivation by insoluble methanol drops results in poor operational stability of the reaction system and unsatisfactory biodiesel yield. The main drawback can be addressed by using solvent engineering. Besides designing and preparing robust biocatalysts, development of effective media for biodiesel production has been receiving extensive attention. Various organic solvents such as tert-butyl alcohol, isooctane, hexane and heptane, and even recently emerging ionic liquids (ILs) have been employed for this purpose. The reason is that they can both dissolve oil (hydrophobic molecules), alcohol, and the byproduct, glycerol (hydrophilic molecules), and make the reaction mixture a homogeneous phase, which is very beneficial to the catalytic performance of the lipases; thus, greatly improving the operational stability and elongating the life span of the lipases. However, every coin has two sides. Solvent-based biodiesel production systems require an additional separation procedure for purification of the product and recovery of the solvent. Table 5
Various lipases used for biodiesel production
Source of lipases
Source of oil
Alcohol
Maximum yields (%)
Burkholderla cepacia Pseudomonas fluorescens Photobacterium lipolyticum Chromoacterlum viscosum Enterobacter aerogenes Bacillus subtilis Candida antarctica Candida sp. 99–125 Candida rugosa Thermomyces lanuginose Rhizopus oryzae Penicillium expansum Geotrichum sp. Rhizomucor miehei Rhizopus chinensis Aspergillus niger
Soy bean oil Soy bean oil Waste oils Jacropha oil Tallow oil Waste cooking oil Olive oil Various oils Rapeseed soapstock Crude palm oil Various oils Corn oil Waste cooking oil Soy bean oil soybean oil Palm oil
Methanol Methanol Methanol Ethanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol
98.0 83.8 70.0 92.0 94.0 90.0 90.0 >87 63.6 96.2 >92 86.0 85.0 92.2 86.0 86.0
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
Biodiesel Production and Technologies
Table 6
267
Commercially available lipases for biodiesel
Type
Source
Marketing company
Fungal lipases
Candida antarctica C. rugosa Mucor miehei Penicillium camembertii Rhizopus oryzae Rhizomucor miehei Thermomyces lanuginosus Pseudomonas cepacia P. fluorescens
Novozymes Sigma, Amano Novozymes Amano Fluka Novozymes Novozymes Amano Amano
Bacterial lipases
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
A novel medium SCF represents an emerging technology for biodiesel production. The SCF-based system combines the extraction and transesterification of the oil, and shows high efficiency. Jackson and King (1996) used immobilized lipases as biocatalysts for transesterification of corn oil in supercritical carbon dioxide (SCCO2). The oils and methanol was pumped in a carbon dioxide stream, respectively. More than 98% fatty acid methyl ester yield was achieved. However, reducing the cost of equipment and medium is the main issue for this technology. In addition, another kind of new solvent, the ILs, can provide a microenvironment where mass transport in SCCO2 can be complemented by the high catalytic efficiency of an enzyme. Thus, biocatalysis in ILs/SCCO2 biphasic systems may be a promising way of providing pure products directly according to design and continuous clean processes. It is concluded that the use of solvent engineering strategy in the enzymatic biodiesel production has the following advantages: (a) less negative effects caused by excessive methanol and glycerol; (b) higher reaction rates and conversion; (c) no regeneration steps for the enzyme reuse; and (d) higher operational stability of the catalyst.
Ultrasonication and microwave assistant treatment The conventional transesterification reaction in batch processing tends to be slow, and phase separation of glycerol is timeconsuming. Ultrasound and microwave, proven to be useful tools in enhancing the reaction rates, have been implemented to overcome the problems. It has been reported that ultrasonic mixing has a significantly positive effect on enzymatic transesterification (Kumar et al., 2011). The higher reaction rates are typically attributed to the enhancement of mass transfer and energy input. In enzymatic catalysis, ultrasonic dispersion can also increase the surface area available to the reactants. Moreover, ultrasonic cavitation increases the efficiency of the catalyst, and optimum conversion can be attained even using a smaller amount of the catalyst. Furthermore, ultrasound as an assistant treatment is often used coupling with other methods to improve the enzymatic transesterification reaction. It has been reported that transesterification reaction assisted by ultrasonic irradiation and vibration reached equilibrium after 4 h, and the biodiesel yield was up to 96 wt%, while the reaction did not reach equilibrium and comparable yield until 12 h by using either ultrasonic or vibration (Yu et al., 2010). Microwave effects in chemical reactions are attributed to the molecular attrition caused by increases of medium temperature and reaction rates. Microwave irradiation has been proven to be a clean, fast, and convenient energy source, and has been widely used in numerous reactions catalyzed by both chemical and biochemical catalysts. In the biodiesel production process, a satisfactory conversion can be achieved in a shorter time under microwave irradiation, when compared to conventional heating (Ros et al., 2012). Moreover, microwave heating is more cost effective than conventional heating. Therefore, the microwave-assisted transesterification reaction is a promising technology in biodiesel production.
Transesterification in CO2 SCF Transesterification in SCFs increases the diffusion rates, which results in higher reaction rates. Of all SCFs, SCCO2 is the most noticeable, because it is nontoxic, nonflammable, cheap, easily available, and of mild critical properties (31.1 C and 7.38 MPa). Another reason is that the favorable transport properties of SCCO2 can accelerate mass-transfer-limited in enzyme-catalyzed reactions. Therefore, most researchers in SCFs have used SCCO2 as a solvent. However, it is reported that the biodiesel yields of enzyme-catalyzed synthesis in SCCO2 were between 40% and 90% and the operational stability of the enzyme was then found to be lower (Oliveira and Rosa, 2006). However, compared with the synthesis in supercritical alcohol, synthesis catalyzed by the enzyme in SCCO2 is carried out at near-ambient temperature. Because the products and the enzyme do not dissolve in carbon dioxide at room temperature, separation can easily be achieved by reducing the pressure (Madras et al., 2004).
Mechanism and kinetics of transesterification The overall process of transesterification reaction is usually considered as a sequence of three continuous reversible steps. In the first step, TG are turned into diglycerides (DG). Then DGs are turned into monoglycerides (MG). Finally, MGs are turned into glycerol. During these three steps of transesterification reaction, fatty acid esters are produced. According to the reaction, 3 mol of alcohol is required to react with 1 mol of TG. An excess amount of alcohol is generally required to drive the reversible reaction to the right side
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Table 7
Several representative examples of microbial production of biodiesel
Biodiesel-producing whole cell Engineered gene
Feedstock
Biodiesel yield
In vitro route (intracellular E. coli E. coli E. coli S. cerevisiae P. pastoris A. oryzae A. oryzae
Vegetable oils, methanol Waste grease, methanol Waste grease, methanol Soybean oil, methanol Waste cooking oil, methanol Rapeseed oil, ethanol Soybean oil, methanol
78–100% 97% 87–95% 71% 82% 94% 98%
Olein, methanol Palm oil, methanol Plant oil hydrolysates, methanol
90% 90% 90%
Soybean oil, methanol Soybean oil, methanol Soybean oil, methanol Soybean oil, methanol Olive oil, methanol
78.3% 83.14% 90% 95.4% 89.4%
Soybean oil, methanol Soybean oil, methanol Waste cooking oil, methanol
95% 95% 87%
LB medium, glucose, sodium oleate LB medium, ethanol
1.28 g L 1 1.99-2.01 mg g
lipase) Proteus sp. lipase Serratia marcescens lipase T. lanuginosus lipase, Candida antarctica lipase B R. oryzae lipase T. lanuginosus lipase Fusarium heterosporum lipase Fusarium heterosporum lipase, Aspergillus oryzae lipase (mdlB) A. oryzae A. oryzae lipase (mdlB) A. oryzae Geobacillus thermocatenulatus lipase A. oryzae C. antarctica lipase B In vitro route (surface displayed lipase) S. cerevisiae R. oryzae lipase P. pastoris Rhizomucor miehei lipase P. pastoris R. miehei lipase, C. antarctica lipase B P. pastoris T. lanuginosus lipase, C. antarctica lipase B E. coli Staphylococcus haemolyticus lipase In vitro route (extracellular lipase) P. pastoris R. oryzae lipase P. pastoris R. miehei lipase, Penicillium cyclopium lipase P. pastoris T. lanuginosus lipase In vitro-in vivo hybrid route E. coli Pdc, Adh, WS/DGAT E. coli Plant thioesterases (ucFatB, ccFatB), WS/DGAT E. coli S. cerevisiae S. cerevisiae In vivo route E. coli E. coli E. coli E. coli
E. coli S. cerevisiae S. cerevisiae
Pdc, Adh, WS/DGAT
LB medium, rice straw hydrolysate/restaurant oil wastes, sodium oleate Overexpression of Gcy1,Dak1,Gup1, and WS/DGAT; Galactose, YNB, glycerol, sodium oleate deletion of GPD2 and FPS1 Candida sp. lipase 2 YPD medium, ethanol Overexpression of Pdc, Adh, accBACD, tesA’, WS/ DGAT, fadD; knockout of fadE Overexpression of Pdc, Adh, accBACD, tesA’,WS/ DGAT (double copies), fadD; knockout of fadE Overexpression of Pdc, Adh, TES, ACL, WS/DGAT, xylanases (xyn10B and xsa); knockout of fadE Overexpression of Pdc, Adh, TES, ACL, WS/DGAT, xylanases and cellulase (gly43F and xyn10B, cel3A and cel); knockout of fadE Overexpression of FAT, FAMT, MAT Acc, wax ester synthase (WS) Disruption of DGA1, LRO1, ARE1, ARE2 and POX1; overexpression of wax ester synthase (WS)
1
0.249-1.27 g L 1
520 mg L 1 11.4 mg g 1
Modified LB medium
922 mg L 1
Minimum medium, glycerol
813 mg L 1
Glucose, xylose, hemicellulose
3.5-674 mg L 1
IL-pretreated switchgrass, xylan/cellobiose, glucose 71-405 mg L 1
M9 minimal medium, glucose SD medium, glucose Glucose, YNB
1.87–22 mM 8.19 mg L 1 17.2 mg L 1
Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
so as to obtain more fatty acid esters. The thermal transesterification reaction can also be thought of the following way: TG reacts with methanol to produce DG which reacts with alcohol to generate MG in the following reaction. At last, MG reacts with alcohol to produce glycerol. At each reaction step, 1 mol of alcohol is consumed while 1 mol of fatty acid ester is produced. This kinetic mechanism can be described by Fig. 3. Kinetics of alcoholysis of triacylglycerols for biodiesel production was usually measured using short chain alcohol as acyl acceptor. The kinetics mechanism for transesterification reaction has been proposed to be represented by Ping-Pong Bi Bi model. For the Ping-Pong Bi Bi with alcohol inhibition, the expression of reaction rate was described by the following equation: ½Strigly½Salcohol Vi ¼ Vmax KMtrigly½Salcoholð1 þ ½Salcohol=KIalcoholÞ þ KMalcohol½Strigly þ ½Strigly½Salcohol
(1)
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Fig. 2 Three types of genetically engineered lipases can be used to mediate in vitro biodiesel production: they can be produced intracellularly by the microbial cell, displayed on its surface or secreted in the medium (oval represents microbial cell used for expressing various forms of lipases). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Table 8
Comparison of different engineered microbial whole cells used for biodiesel production
Biodiesel-producing microbe
Expression level
In vitro route (intracellular lipase) E. coli Low S. cerevisiae Moderate P. pastoris High A. oryzae High In vitro route (extracellular lipase) P. pastoris High In vitro route (surface displayed lipase) S. cerevisiae Low P. pastoris Low E. coli Low Ex vivo route E. coli Low S. cerevisiae Low In vivo route E. coli Low S. cerevisiae Low
Cell growth
Cell mass
Immobilization
Substrate transfer
Reaction time
Biodiesel yield
Hours to days Days to one week Days to one week Days to one week
Low Moderate High Moderate
Not required Not required Not required Required
Difficult Difficult Difficult Difficult
Moderate Long Long Long
High Moderate Moderate High
Days to one week
High
Not required
Easy
Short
High
Days to one week Days to one week Hours to days
Moderate High Low
Not required Not required Not required
Easy Easy Easy
Short Short Short
High High High
Hours to days Days to one week
Low Moderate
Not required Not required
Moderate Moderate
Moderate Moderate
Low Low
Hours to days Days to one week
Low Moderate
Not required Not required
Moderate Moderate
Moderate Moderate
Low Low
Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 3
Three steps of reversible transesterification reaction.
where, Vi stands for the initial reaction rate, Vmax stands for the maximum reaction rate, KMtrigly stands for the Michaelis–Menten constant for TGs, and KMalcohol is the Michaelis–Menten constant for alcohol. KIalcohol stands for the inhibition constant for alcohol. Strigly and Salcohol stand for the initial concentration of TGs and alcohol, respectively. The Ping-Pong Bi Bi mechanism could also have been used in the kinetic studies of esterifications of long-chain fatty acids catalyzed by lipases. It can be described as the following steps: (a) as the nucleophile, the oxygen in the OeH group of lipase is added to generate enzyme–substrate complex; (b) first, proton is located in the conjugate acid of the amine, then, it moves to the alkyl oxygen atom of the substrate, resulting in a glycerol moiety. If the initial reaction substrate was TG, then a DG will be generated. Similarly, if the substrate is DG, then MG will be generated; (c) In the methanol molecule, the oxygen atom is added to the acyl enzyme intermediate, which contains the carbon atom of the C]O. Then, the complex, which consists of acylated enzyme–alcohol, will be formed; (d) the enzyme oxygen atom is removed from the complex, and a proton migrates from the conjugate acid of the amine, generating FAME (Al-Zuhair et al., 2007).
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If the alcoholysis of triacylglycerols for biodiesel production is only inhibited by alcohol, the Ping-Pong kinetic model with competitive inhibition can be described by the following equation: V¼
V h max i 1 þ ½KAA 1 þ K½BiB þ ½KBB
(2)
where, V stands for the initial rate of reaction, Vmax stands for the maximum rate of reaction, KA stands for the binding constants of the fatty acid (A) and KB stands for the binding constants of the alcohol (B), and KiB stands for the inhibition constant of the alcohol. The studies of kinetics and the mechanism of enzymatic-catalyzed transesterification reaction can provide the essential parameters for the selection of the best lipase system in biodiesel production. However, the mechanisms of transesterification reaction for biodiesel production by lipase catalysis have not yet been thoroughly studied, thus none of the reported explanations has been widely accepted unambiguously. The complete explanations of the mechanism on the transesterification alcoholysis of triacylglycerols for biodiesel production need further, deeper investigation.
Engineering of Microorganism to Biosynthesize Biodiesel In addition to in vitro lipase mediated biodiesel production, genetic engineering and metabolic engineering of a chassis like E. coli and S. cerevisiae to produce biodiesel based on genetically modifying and system regulation of the fatty acid biosynthetic pathway or/and introducing heterologous or homologous ethanol biosynthetic pathway has been recently emerging (Table 8). The chassis can be metabolically redesigned and systematically regulated to create a balanced biodiesel-producing microbial cell factory (Figs. 4 and 5). Fatty acid biosynthesis and the degradation pathway are well elucidated in model microorganism E. coli. Fatty acyl is a central
Fig. 4 Schematic representation of microbial production of biodiesel via in vitro (A), ex vivo (B and C) and in vivo (D) routes. Depending on each case, the substrates (feedstock) can be provided exogenously or synthetized by the microbial cell. The microbial enzymes required, including lipases and other biodiesel biosynthetic enzymes, are indicated inside the cells (oval shapes). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 5 A dynamic sensor-regulator system containing the repressor gene fadR and FadR-regulated promoters (PmodB and PmodC) for enhancing FAEE titer via bio-sensing intermediate fatty acyl-CoA. Adapted from Zhang et al., 2012.
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metabolite used as a precursor for multiple fuels and chemicals. Fatty acid ethyl ester as a fatty acid-derived product can be de novo biosynthesized by engineering of fatty acid biosynthetic and degradation pathways. Overexpression of key enzymes involved in synthesis steps and knocking out the critical enzymes responsible for fatty acid degradation can accumulate fatty acyl-CoA. Addition of exgenous ethanol or introducing an ethanol formation pathway could confer the host strain fatty acid ethyl ester-producing capacity. Under optimized fed-batch microbial fermentation, an engineered E. coli produced biodiesel with a titer of 922 mg L 1 fatty acid ethyl esters (Duan et al., 2011). Especially, using a genetically tractable oleaginous host such as Y. lipolytica as a chassis can supply a sufficient fatty acid pool, which would significantly increase the biodiesel yield. Currently, we are developing oleaginous Y. lipolytica yeast as a cell factory for biodiesel production according to the route described in Figs. 6 and 7.
Fig. 6 Proposed routes for metabolic engineering of oleaginous Y. lipolytica yeast for biosynthesis of biodiesel based on lipase (dotted line and enzyme in blue represent introduced pathway, enzymes colored in green require overexpression, represents knocked out enzymes, red intermediates represent central metabolites in this engineered pathway). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 7 Proposed routes for metabolic engineering of oleaginous Y. lipolytica yeast for biosynthesis of biodiesel based on WS (dotted line and enzyme in blue represent introduced pathway, enzymes colored in green require overexpression, represents knocked out enzymes, red intermediates represent central metabolites in this engineered pathway). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
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Prospect and Conclusion Although alkali-catalyzed biodiesel production has already achieved commercialization, in vitro enzymatic conversion of costeffective feedstock to biodiesel based on liquid free lipases, various immobilized lipases, and whole cell lipase biocatalysts would come to be maintream technologies due to their greener and cleaner properties. Significantly, in vivo de novo biosynthesis of biodiesel from glucose, glycerol, and even lignincellulosic biomass derived sugars, represents the frontier of biodiesel production. Especially, the combination of genentic engineering, metabolic engineering, systems biology, and synthetic biology technologies will advance the biodiesel-producing microbial cell factory from laboratory scale to industrial scale in the near future. To make biodiesel production more economical and competitive, low cost oils, including waste cooking oils, high acid value oils, forestry oils, and microbial oils, are primary options. From the perspective of environmentally friendly biodiesel manufacturing, enzymatic transformation of cost-effective oils to biodiesel via a variety of immobilized lipases and whole cell biocatalysts should be seriously considered and thoroughly investigated. Furthermore, redesigning and constructing a biodieselproducing microbial cell factory from lignincellulosic biomass derived sugars is a promising technology that is worth developing.
References Al-Zuhair, S., Ling, F.W., Jun, L.S., 2007. Proposed kinetic mechanism of the production of biodiesel from palm oil using lipase. Process Biochemistry 42, 951–960. Calvin, M., 1985. Fuel oils from higher plants. In: Annual Proceedings of the Phytochemical Society of Europe, 26, pp. 147–160. Duan, Y., Zhu, Z., Cai, K., Tan, X., Lv, X., 2011. De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation. Plos One 6, 1–7. Goering, C.E., Camppion, R.N., Schwab, A.W., Pryde, E.H., 1982. Vegetable oil fuels. In: International Conference on Plant and Vegetable Oils as FuelsAmerican Society of Agricultural Engineers, St. Joseph, MI, pp. 279–286. Fargo, North Dakota. Jackson, M.A., King, J.W., 1996. Methanolysis of seed oils in flowing supercritical carbon dioxide. Journal of the American Oil Chemists Society 73, 353–356. Krawczyk, T., 1996. Biodiesel-alternative fuel makes inroads but hurdles remain. Inform 7, 801–829. Kumar, G., Kumar, D., Poonam, Johari, R., Singh, C.P., 2011. Enzymatic transesterification of Jatropha curcas oil assisted by ultrasonication. Ultrasonics Sonochemistry 18, 923–927. Luque, R., Herrero-Davila, L., Campelo, J.M., Clark, J.H., Hidalgo, J.M., Luna, D., 2008. Biofuels: A technological perspective. Energy & Environmental Science 1, 513–596. Ma, F., Hanna, M.A., 1999. Biodiesel production: A review. Bioresource Technology 70, 1–15. Ma, F., Hanna, M.A., 1999. Biodiesel production: A review. Bioresource Technology 70, 1–15. Madras, G., Kolluru, C., Kumar, R., 2004. Synthesis of biodiesel in supercritical fluids. Fuel 83, 2029–2033. Nagel, N., Lemke, P., 1990. Production of methyl fuel from miceoalgea. Applied Biochemistry and Biotechnology 24, 355–361. Oliveira, A.C., Rosa, M.F., 2006. Enzymatic transesterification of sunflower oil in an aqueous-oil biphasic system. Journal of the American Oil Chemists Society 83, 21–25. Rachmaniah, O., Ju, Y.H., Vali, S.R., Tjondronegoro, I., Musfil, A.S., 2004. A study on acid-catalyzed transesterification of crude rice bran oil for biodiesel production. In: Youth Energy Symposium on 19th World Energy Congress and Exhibition. September 5–9. Sydney, Australia. Ros, P.C., Castro, H.F., Carvalho, A.K., Soares, C.M., Moraes, F.F., Zanin, G.M., 2012. Microwave-assisted enzymatic synthesis of beef tallow biodiesel. Journal of Industrial Microbiology & Biotechnology 39, 529–536. Yu, D., Tian, L., Wu, H., Wang, S., Wang, Y., Ma, D., Fang, X., 2010. Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435. Process Biochemistry 45, 519–525. Ziejewski, M., Kaufman, K.R., Schwab, A.W., Pryde, E.H., 1984. Diesel engine evaluation of a nonionic sunflower oil-aqueous ethanol microemulsion. Journal of the American Oil Chemists Society 61, 1620–1626.
Introduction As an alternative to fossil fuels, green and clean biofuels must be technically feasible, economically competitive, and environmentally acceptable. One of them, biodiesel, an alternative diesel fuel, is made from renewable sources of oils and fats. It is biodegradable and nontoxic, with a low emission profile, so it is environmentally beneficial. Biodiesel is a mixture of mono-alkyl esters produced from vegetable oils, animal fats, forestry oils, waste cooking oils, algae oils, etc. In recent decades, biodiesel has been drawing wide attention for its ability to replace fossil fuels, which are likely to be exhausted in the near future. Additionally, the environmental issues caused by emission of gases from combustion of fossil fuels have also encouraged development of biodiesel, which is considered to be a carbon neutral fuel, because its carbon content is originally fixed from the atmosphere. In general, to develop biodiesel from biological resources is of great significance to the development of mankind. One hundred years ago, Rudolf Diesel tested vegetable oil as fuel. With the advent of cheap petroleum, appropriate crude oil fractions were refined to serve as fuel, and diesel fuels and diesel engines evolved together. In the 1930s to 1940s, vegetable oils were used as diesel fuels usually only in emergency situations. Recently, because of increases in crude oil prices, limited resources of fossil oils, and environmental concerns, there has been a renewed focus on plant oils and animal fats to make biodiesel fuels. Continuing and increasing use of petroleum will intensify local air pollution and magnify the global warming problems caused by CO2. In a particular case, such as the emission of pollutants in the closed environments of underground mines, biodiesel fuel has the potential to reduce the level of pollutants and the level of potential or probable carcinogens (Krawczyk, 1996). Fats and oils are primarily water-insoluble, hydrophobic substances in the plant and animal kingdoms that are made up of one mole of glycerol and three moles of fatty acids (commonly referred to as triglycerides, TGs). Fatty acids vary in carbon chain length and in the number of unsaturated bonds (double bonds). For example, in beef tallow, the saturated fatty acid component accounts for almost 50% of the total fatty acids, while in soybean oil, the unsaturated fatty acid component accounts for almost 85%. However, the higher stearic and palmitic acid contents give beef tallow the unique properties of a higher melting point and higher viscosity compared to soybean oil. Table 1 shows typical fatty acid compositions of common oil sources. As is known, the kinematic viscosity of oils and fats is much higher than that of diesel, owing to their higher molecular weight and complex structure. In order to reduce viscosity of oils and fats, alcohol is usually employed to replace glycerol via transesterification reaction. Therefore, biodiesel can be synthesized by chemically combining any natural oils or fats with an alcohol such as methanol or ethanol by the catalysis of an alkali/acid or a lipase (see Fig. 1). Methanol and ethanol are the most commonly used alcohols in the commercial production of biodiesel. Though biodiesels are esters of natural oils, different oils retain similar chemical and physical properties when they are transesterified. So far, abundant research has proven that biodiesel made from plant oils and animal fats can be effectively used in diesel engines without any modification. Moreover, the energy density of biodiesel is very close to fossil diesel. Similar combustion properties between biodiesel and petroleum-derived diesel have made the former one of the most promising renewable and sustainable fuels for the automobile.
Feedstock of Biodiesel Vegetable oils and animal fats are extracted, or pressed, to get crude oil or fat. These usually contain free fatty acids, phospholipids, sterols, water, odorants, and other impurities. Even the refined oils and fats also retain small amounts of free fatty acids and water. Table 1
Typical fatty acid compositions of common oil sources
Oils
C16:0
Corn Rapeseed Soybean Peanut Sesame Rice bran Wheat grain Sunflower Groundnut Crambe Linseed Safflower Cottonseed
11.7 3.5 11.4 11.4 13.0 11.7–16.5 20.6 7.1 8.5 2.1 5.0 7.3 28.3
C16:1
C18:0
C18:1
C18:2
C18:3
C20:0
25.2 64.4 20.8 48.3 53.0 39.2–43.7 16.6 25.5 51.6 18.9 20.0 13.5 13.3
60.6 22.3 53.8 32.0 30.0 26.4–35.1 56.0 62.4 26.0 9.0 18.0 77.0 57.5
0.5 8.2 9.3 0.9
0.2
1.0
1.9 0.9 4.4 2.4 4.0 1.7–20.5 1.1 4.7 6.0 0.7 2.0 1.9 0.9
0.1
Encyclopedia of Sustainable Technologies, Volume 3
0.3 1.3
C20:1
C22:0
C22:1
2.5
n9
C24:0
Others
1.2
0.4–0.6
0.4–0.9
2.9
1.8 0.3
6.9 55.0
2.1
http://dx.doi.org/10.1016/B978-0-12-409548-9.10110-1
0.8
58.5
1.1 0.2
261
262
Fig. 1
Biodiesel Production and Technologies
Formula of biodiesel production with short chain alcohols as acyl acceptors.
The free fatty acid and water contents have significant effects on the transesterification reaction of glycerides with alcohols ether using alkaline or acid as the catalyst. They also make the procedure of separating fatty acid esters and glycerol complex and very difficult. Considerable research has been done on using plant oils, especially vegetable oils, including palm oil, soybean oil, sunflower oil, coconut oil, rapeseed oil, tung oil, etc., as diesel fuel. Animal fats, although mentioned frequently, have not been studied to the same extent as vegetable oils. Some methods applied to vegetable oils are not applicable to animal fats because of physiochemical property differences. Recently, oils from algae, bacteria, and fungi have also been investigated. Microalgae, in particular, can grow very fast, resulting in a very short life cycle, but with very high oil content. They are being regarded as an ideal resource as biodiesel feedstock. Oils from microalgae have been examined as a source of methyl ester diesel fuel (Nagel and Lemke, 1990). In addition, terpenes and latexes are occasionally studied as diesel fuels (Calvin, 1985). An important reason to screen feedstock is that the biodiesel production cost is mainly dependent on feedstock price. Despite the widespread use of vegetable oils such as soybean oils and rapeseed oils for production of biodiesel, increasing competition between the production of biofuels and food has limited further application on an industrial scale, especially in developing countries. In this context, use of cost-effective oil resources replacing edible oils for biodiesel production is more economically viable. Waste cooking oils - abundant and cheap wastes from urban catering - have been widely used for synthesis of biodiesel. However, waste cooking oils generally contain large amount of free fatty acids. Therefore, during conversion of waste cooking oils into biodiesel, transesterification of triacylglycerols and esterification of free fatty acids are both performed. Alkali-catalyzed biodiesel production from waste cooking oils is not technically feasible without neutralization in the pretreatment step, because the saponification reaction between alkali catalyst and free fatty acid would limit a further conversion process. Lipase is feasible to convert these oils containing high free fatty acid into biodiesel because of its ability to simultaneously catalyze transesterification and esterification reactions. Other nonedible oils suitable for biodiesel production are summarized in Table 2. Microorganism derived oils (also called single cell oils), generated by oleaginous yeast, fungi, bacteria, and microalgae, are regarded as emerging feedstock for biodiesel production (Table 3). These oleaginous microorganisms generally accumulate lipids in the main form of triacylglycerols accounting to more than 20% of its dry cell biomass. Yarrowia lipolytica is a well known superior oleaginous yeast, which can generate than 30% of its dry cell weight in oil. Moreover, Y. lipolytica yeast is generally recognized as safe, capable of growing well on low cost substrates such as residues from agriculture and industry. Even better, the lipid biosynthetic pathway of the yeast can be genetically modified for enhancing lipid production by metabolic engineering and reconstruction. Thus, Y. lipolytica yeast has been used as a chassis for constructing cell factories for oil production.
Technologies for Biodiesel Production There are three major conversion technologies for biodiesel production: acid/base catalysis, enzyme catalysis, and supercritical fluid (SCF) technology. Acid/base catalysis has high requirements for water content and acid value of raw materials, and certain pollutants are produced. Enzyme catalysis takes a relatively long time, and the cost of the enzyme is rather high. The SCF method has recently emerged as a method of biodiesel production, but the technology is still in its infancy, and the process is expensive and complex. Therefore, development of green catalysts and ideal technologies will take time. However, technologies for biodiesel have been in development for a long time.
Direct Use and Blending The properties of heat content (80% of diesel fuel), availability, and renewability, make vegetable oils a potential alternative for diesel fuel. It is not recommended to use 100% oil for diesel fuel, because of its high viscosity. People found that this problem can be minimized by blending vegetable oils with pure diesel. Using a vegetable oils/diesel fuel blend is the simplest means to reduce the viscosity of vegetable oil, with no need for any chemical procedure. So far, blends of conventional diesel fuel with sunflower oil, soybean oil, cottonseed oil, rapeseed oil, Jatropha curcas oil, and Pongamia pinnata oil have been reported. Blending waste cooking oils and diesel with a suitable ratio has also been applied. A mixture of 20% vegetable oil and 80% diesel fuel was confirmed feasible, and some experiments even ran up to 50/50. However, obvious disadvantages, such as higher viscosity, lower volatility, and the reactivity of unsaturated hydrocarbon chains, limit widespread application. Thus, this blend was considered not suitable for long-term use in a direct injection engine.
Biodiesel Production and Technologies
Table 2
263
Biodiesel production from feedstock of nonedible oils
Nonedible oils
Genus/species
Distribution
Balanos oil Bladderpod oil
Balanites aegyptiaca Physaria
Castor oil Chaulmoogra oil Copaiba Crambe oil Croton oil Cuphea oil Honesty oil Honge oil Illipe butter Jatropha oil Jojoba oil
Castor beans Hydnocarpus wightiana Copaifera Crambe abyssinica Croton tiglium Cuphea Lunaria annua Millettia pinnata Shorea stenoptera Euphorbiaceae Simmondsia chinensis
Mahua Mango oil Milk bush Nahor oil Neem oil Petroleum nut oil Rubber seed oil Sea buckthorn oil Stillingia oil Tall oil Tamanu Tonka bean oil Tung oil Vernonia oil
Madhuca indica Mangifera indica Euphorbia tirucalli Mesua ferrea L. Azadirachta indica Pittosporum resiniferum Rubber tree Hippophae rhamnoides Sapium sebiferum wood pulp manufacture Calophyllum inophyllum Dipteryx odorata Vernicia fordii Vernonia galamensis
Egypt Southwestern United States, eastward to Kansas and southward into northern Mexico Mediterranean Basin, Eastern Africa, and India Indonesia, Malaysia, and the Philippines Tropical Africa and North and South American Europe, southwest and central Asia, and eastern Africa India and the Malay Archipelago America South east Europe and western Asia Asia Southeast Asia, Borneo, and Sumatra India Southern Arizona, Southern California, and northwestern Mexico India India Africa Indian origin Indian subcontinent and other areas in the tropics Philippines Cambodia and Nigeria Europe and Asia China India and Nigeria Southeast Asia Northern South America China East Africa
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and, Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
Microemulsification of Oils Microemulsification is a method for forming microemulsions, which are transparent, thermodynamically stable colloidal dispersions. To solve the problem of the high viscosity of vegetable oils, microemulsions with immiscible liquids, such as methanol, ethanol, and ionic or nonionic amphiphiles have been investigated. It has been proven that short-term performances of both ionic and nonionic microemulsions of aqueous ethanol in soybean oil are comparable to that of No. 2 diesel fuel (Goering, et al., 1982). Ziejewski et al. (1984) conducted a microemulsion with 53% (v/v) alkali-refined and winterized sunflower oil, 13.3% (v/v) 190proof ethanol, and 33.4% (v/v) 1-butanol. This microemulsion possessed a viscosity of 6.31 cSt at 313 K, a cetane number of 25, and an ash content less than 0.01%. Lower viscosities and better spray patterns were observed with an increase of 1-butanol.
Thermal Cracking (Pyrolysis) Producing biofuels from vegetable oils by thermal cracking (pyrolysis) is a promising alternative. The purpose of pyrolysis is to optimize high-value fuel products from biomass through thermal means or coupling with catalysts. The potential materials can be any type of biomass, such as vegetable oils, animal fats, wood, biowaste, etc. Many studies have reported the pyrolysis of TGs to obtain products suitable for diesel engines. Thermal pyrolysis of TGs produces many compounds, including alkanes, alkenes, alkadienes, aromatics, and carboxylic acids. Different types of vegetable oils lead to significant differences in the composition of the pyrolyzed oil. The mechanism for the thermal decomposition of TGs is complex, owing to the structures and multiplicity of possible reactions of the mixed TGs. In general, pyrolysis of these structures occurs via either a free-radical or carbonium ion mechanism. Formation of aromatics is supposed by a Diels-Alder addition of ethylene to a conjugated diene formed in the pyrolysis reaction. The carboxylic acids formed during the pyrolysis of vegetable oils probably result from cleavage of the glyceride moiety. Until recently, several vegetable oils, such as palm oil, canola oil, and soybean oil, have been employed in the process involving conversion of the oils into biofuels suitable for gasoline engines using acid catalysts (transition metal catalysts which yield biofuels enriched in diesel fractiondover 50% by weight, and zeolites and mesoporous materials that give biofuels with higher gasoline fractionsdover 40%, with higher aromatic content) (Luque, et al., 2008). The reaction is usually conducted at moderate to high temperatures
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Table 3
Oil content of microorganisms
Microorganisms Microalgae Botryococcus braunii Chlorella sorokiana C. emersonii C. vulgaris Crypthecodium cohnii Cylindrotheca sp. Dunaliella salina Isochrysis galbana Monodus subterraneus Nannochloropsis oculata Neochloris oleoabundans Nitzschia sp. N. laevis Phaeodactylum tricornutum Porphyridium cruentum Parietochloris incise Scenedesmus dimorphus S. obliquus Schizochytrium sp. Spirulina platensis Bacterium Arthrobacter sp. Acinetobacter calcoaceticus Bacillus alcalophilus Rhodococcus opacus Yeast Candida curvata Candida sp. 107 C. valida C. utilis C. curvatus Cryptococcus albidus Lipomyces starkeyi Rhodotorula glutinis R. graminis Rhizopus arrhizus Rhodosporidium toruloides Trichosporon pullulans Yarrowia Lipolytica Fungi Aspergillus oryzae Cunninghamella japonica C. bainieri Humicola lanuginose Mucor circinelloides M. rouxii Mortierella isabellina M. vinacea M. ramanniana
Oil content (% dry cell weight)
25–75 22 29.63 56.6 56 16–37 14–20 14.5 39.3 22–44 35–65 45–47 69.1 6–7 19.3 62 16–40 35–55 50–77 5–17 >40 27–38 18–40 24–25 58 42 26–37 64 72 65 63 72 36 57 48–67.5 65 36 57 50 38 75 23 32 86 66 54.2
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
(573–773 K) using different oils/catalyst ratios, depending on the oil and the catalyst. Catalytic cracking not only increases the yield of gasoline by breaking large molecules into smaller ones, but also improves the quality of the gasoline: this process involves carbocations and yields alkanes and alkenes with the highly branched structures desirable in gasoline. The main components are alkanes and alkenes, which account for 60% of the total weight. Carboxylic acids account for another 9.6%–16.1% (Ma and Hanna, 1999a, 1999b). However, the heterogeneous products and high temperature are the main drawbacks for cracking oils into diesel target product. It is also difficult to control selectivity of cracking products.
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Transesterification (Alcoholysis) Transesterification reaction is the use of a kind of oil and an alcohol as substrates to prepare esters and glycerol. The reversible reaction process is also called alcoholysis. To drive the equilibrium to the product direction, excess alcohol is often loaded. Various oilrich feedstocks, including vegetable oils, waste oils, and microalge or microorganism derived oils, and methanol or ethanol are frequently used for biodiesel production. A catalyst responsible for improving the reaction rate and biodiesel yield can be alkalis, acids, or enzymes.
Transesterification catalyzed by chemical catalysts As for chemical-catalyzed biodiesel production via transesterification reaction, NaOH, KOH, carbonates and corresponding sodium and potassium alkoxides, such as sodium methoxide, are frequently served as alkali catalysts. Sulfuric acid, sulfonic acids, and hydrochloric acid are usual acid catalysts. Various chemical catalyses for biodiesel production on reactors are summarized in Table 4.
Acid-catalytic transesterification methods Sulfuric, phosphoric, hydrochloric, and organic sulfonic acids can be used to catalyze transesterification reaction to produce biodiesel. Currently, the more-used catalysts in biodiesel production are organic acids: for example, the derivates of toluenesulfonic acid and, more often, mineral acids such as sulfuric acid. Usually, the transesterification rate if using acid catalysts is much slower than that with alkali catalysis. But if high contents of water and FFAs are present in the vegetable oil, acid-catalyzed transesterification can be used. The acid-catalyzed reaction commonly needs high temperatures beyond 373 K and reaction times of 3–48 h. These catalysts give fairly high yields in the transesterification process. Acid-catalyzed reactions also require the use of high alcohol-to-oil molar ratios in order to obtain ideal product yields in the practical reaction. However, ester yields do not proportionally increase with molar ratio. Rachmaniah et al. (2004) investigated transesterification of the refined soybean oil (> 99% TGs) and low grade high FFA (60%) rice bran oil with methanol by using 10% hydrochloric acid as a catalyst (w/w) at a temperature of 343 K. In their study, all reactions were conducted with a methanol molar ratio of 20:1. The highest FAME content of 90% was obtained from transesterification of low grade high FFA rice bran oil with a reaction time of 6 h.
Alkali-catalytic transesterification methods Alkali-catalytic transesterification is catalyzed by alkaline metal alkoxides and hydroxides, as well as sodium or potassium carbonates. Sodium methoxide is the most widely used biodiesel catalyst. Alkaline catalysts have the following advantages: (a) short reaction time; (b) relatively low temperature; (c) only a small amount of catalyst used; and (d) little or no darkening of color of the oil. Nowadays, among commercialization technologies of biodiesel production, alkali-catalyzed transesterification is the main feasible route because of the above merits. However, for the oils containing significant amounts of FFAs, alkaline catalysts do not convert it to biodiesel, but rather into a lot of soap. These FFAs react with the alkaline catalyst to produce soaps that inhibit the separation of the biodiesel, glycerin, and wash water. In alkali-catalytic transesterification, the catalyst is first dissolved into alcohol by vigorous stirring, and then the catalyst/alcohol mixture is pumped into oil to react and stirred vigorously for 2 h at 340 K in ambient pressure. A successful transesterification reaction produces two liquid phases: ester and crude glycerol. Generally, the alkali-catalyzed transesterification process is conducted at low temperatures and pressures (333–338 K and 1.4–4.2 bar) with low catalyst concentrations (0.5–2 wt%), and a conversion rate of over 95% can be expected after 1 h reaction. Although the homogeneous-catalyzed biodiesel reaction is relatively fast and results in high conversions, it has some serious drawbacks: (a) the catalyst cannot be recovered; (b) it must be neutralized and separated from the methyl ester phase at the end of the reaction; and (c) it consequently generates a large volume of wastewater. The above problems have provided an impetus to search for new stable and more environmentally friendly solid catalysts. For searches of promising solid base catalysts, attempts were made with alkali earth oxides, such as CaO, SrO, and BaO. Alkali and alkali earth salts loaded on metal oxide such as KOH/ Al2O3, KF/MgO, Sr(NO)3/ZnO, Ca(NO)3/Al2O3, and calcined Mg–Al hydrotalcites. On the other hand, a variety of solid acids has already been tested, including carbon-based solid acid, WO3/ZrO2, SO4/ZrO2, SO4/TiO2, SO4/ZrO2-Al2O3, and Nafion. They can Table 4
Representative chemical catalysis for biodiesel production on reactors
Reactor type
Feedstock
Catalyst
FAME yield (%)
Membrane reactor Zigzag micro-channel reactor Oscillatory flow reactor Trickle bed reactor rotating packed bed Stirred-tank reactor Reactive distillation reactor
Canola oil, methanol Soybean oil, methanol Rapeseed oil, methanol Rapeseed oil, methanol soybean oil methanol Palm oil, methanol Canola oil, methanol
NaOH, H2SO4 NaOH NaOH Alkaline KOH KOH KOH
96, 64 81.5–99.5 99 95 97.3 58.5–97.3 94.4
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
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obtain catalytic efficiency similar to those of homogeneous catalysts. What’s more, they endow the catalyst recovery property. Nevertheless, the chemical route for biodiesel production generally requires higher energy input than the enzymatic route.
Transesterification catalyzed by lipases Although the alkali-catalytic transesterification process gives high conversion of TGs to biodiesel in short reaction times, it suffers from several shortcomings: (a) it is energy intensive; (b) the recovery of glycerol is difficult; (c) the alkaline catalyst has to be removed from the product; (d) alkaline wastewater requires treatment; and (e) FFAs and water interfere with the reaction. Enzymatic catalysts like lipases are able to effectively catalyze the transesterification of TGs in either aqueous or nonaqueous systems. Thus, they can overcome the above-mentioned problems. Particularly, the byproduct, glycerol, can be easily recovered with a simple separation process. However, enzymatic catalysts are often more expensive than chemical catalysts, so recycling and reusing strategies are often applied for commercial viability. Though the high cost of enzyme production is a major obstacle to the commercialization of enzyme-catalyzed processes, recent advances in enzyme technology, such as use of solvent-tolerant lipases (especially immobilized lipases), making catalyst reutilization possible, has developed cost-effective systems. It can be seen that enzymatic biodiesel production by lipase catalyzed transesterification is a clean and green route. An enzymatic route with mild reaction conditions and a stoichiometrical amount of alcohol dosage show obvious advantages over chemcal routes. Especially, simultaneous esterification of free fatty acid and transesterification with triacylglycerol for biodiesel production can be achieved by lipase mediated biotransformation. Various lipases from bacteria such as Burkholderia cepacia, yeast such as Candida antarctica, and filamentous fungus such as Thermomyces lanuginosus have often been used for this process (Table 5). Several lipases for biodiesel production are commercially available (Table 6). Biocatalyst cost is one of the main factors affecting the overall cost of biodoesel production. Overexpression of desirable lipases in genetically tractable hosts such as E. coli, Bacillus subtilis, and Pichia pastoris is an effective strategy to lower the biocatalyst cost. Additionally, immobilization of lipases can further reduce the biocatalyst cost due to improved tolerance to solvents and recyclability. Various materials and technologies have been developed for immobilization of lipases, and great progress has been made. Alternatively, lipases in the forms of whole cells, including intracellular type, surface displayed type, and extracellular type, have been widely used to catalyze biodiesel production (Table 7, Fig. 2). Advantage and drawback comparison of different engineered microbial whole cells used for biodiesel production is summarized in Table 8.
Transesterification via solvent engineering Although biodiesel production in solvent-free systems is extensively reported, lipase inactivation by insoluble methanol drops results in poor operational stability of the reaction system and unsatisfactory biodiesel yield. The main drawback can be addressed by using solvent engineering. Besides designing and preparing robust biocatalysts, development of effective media for biodiesel production has been receiving extensive attention. Various organic solvents such as tert-butyl alcohol, isooctane, hexane and heptane, and even recently emerging ionic liquids (ILs) have been employed for this purpose. The reason is that they can both dissolve oil (hydrophobic molecules), alcohol, and the byproduct, glycerol (hydrophilic molecules), and make the reaction mixture a homogeneous phase, which is very beneficial to the catalytic performance of the lipases; thus, greatly improving the operational stability and elongating the life span of the lipases. However, every coin has two sides. Solvent-based biodiesel production systems require an additional separation procedure for purification of the product and recovery of the solvent. Table 5
Various lipases used for biodiesel production
Source of lipases
Source of oil
Alcohol
Maximum yields (%)
Burkholderla cepacia Pseudomonas fluorescens Photobacterium lipolyticum Chromoacterlum viscosum Enterobacter aerogenes Bacillus subtilis Candida antarctica Candida sp. 99–125 Candida rugosa Thermomyces lanuginose Rhizopus oryzae Penicillium expansum Geotrichum sp. Rhizomucor miehei Rhizopus chinensis Aspergillus niger
Soy bean oil Soy bean oil Waste oils Jacropha oil Tallow oil Waste cooking oil Olive oil Various oils Rapeseed soapstock Crude palm oil Various oils Corn oil Waste cooking oil Soy bean oil soybean oil Palm oil
Methanol Methanol Methanol Ethanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol
98.0 83.8 70.0 92.0 94.0 90.0 90.0 >87 63.6 96.2 >92 86.0 85.0 92.2 86.0 86.0
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
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Table 6
267
Commercially available lipases for biodiesel
Type
Source
Marketing company
Fungal lipases
Candida antarctica C. rugosa Mucor miehei Penicillium camembertii Rhizopus oryzae Rhizomucor miehei Thermomyces lanuginosus Pseudomonas cepacia P. fluorescens
Novozymes Sigma, Amano Novozymes Amano Fluka Novozymes Novozymes Amano Amano
Bacterial lipases
Adapted from Yan, Y., Li, X., Wang, G., Gui, X., Li, G., Su, F., Wang, X. and Liu, T. (2014). Biotechnological preparation of biodiesel and its high-valued derivatives: A review. Applied Energy 113, 1614–1631.
A novel medium SCF represents an emerging technology for biodiesel production. The SCF-based system combines the extraction and transesterification of the oil, and shows high efficiency. Jackson and King (1996) used immobilized lipases as biocatalysts for transesterification of corn oil in supercritical carbon dioxide (SCCO2). The oils and methanol was pumped in a carbon dioxide stream, respectively. More than 98% fatty acid methyl ester yield was achieved. However, reducing the cost of equipment and medium is the main issue for this technology. In addition, another kind of new solvent, the ILs, can provide a microenvironment where mass transport in SCCO2 can be complemented by the high catalytic efficiency of an enzyme. Thus, biocatalysis in ILs/SCCO2 biphasic systems may be a promising way of providing pure products directly according to design and continuous clean processes. It is concluded that the use of solvent engineering strategy in the enzymatic biodiesel production has the following advantages: (a) less negative effects caused by excessive methanol and glycerol; (b) higher reaction rates and conversion; (c) no regeneration steps for the enzyme reuse; and (d) higher operational stability of the catalyst.
Ultrasonication and microwave assistant treatment The conventional transesterification reaction in batch processing tends to be slow, and phase separation of glycerol is timeconsuming. Ultrasound and microwave, proven to be useful tools in enhancing the reaction rates, have been implemented to overcome the problems. It has been reported that ultrasonic mixing has a significantly positive effect on enzymatic transesterification (Kumar et al., 2011). The higher reaction rates are typically attributed to the enhancement of mass transfer and energy input. In enzymatic catalysis, ultrasonic dispersion can also increase the surface area available to the reactants. Moreover, ultrasonic cavitation increases the efficiency of the catalyst, and optimum conversion can be attained even using a smaller amount of the catalyst. Furthermore, ultrasound as an assistant treatment is often used coupling with other methods to improve the enzymatic transesterification reaction. It has been reported that transesterification reaction assisted by ultrasonic irradiation and vibration reached equilibrium after 4 h, and the biodiesel yield was up to 96 wt%, while the reaction did not reach equilibrium and comparable yield until 12 h by using either ultrasonic or vibration (Yu et al., 2010). Microwave effects in chemical reactions are attributed to the molecular attrition caused by increases of medium temperature and reaction rates. Microwave irradiation has been proven to be a clean, fast, and convenient energy source, and has been widely used in numerous reactions catalyzed by both chemical and biochemical catalysts. In the biodiesel production process, a satisfactory conversion can be achieved in a shorter time under microwave irradiation, when compared to conventional heating (Ros et al., 2012). Moreover, microwave heating is more cost effective than conventional heating. Therefore, the microwave-assisted transesterification reaction is a promising technology in biodiesel production.
Transesterification in CO2 SCF Transesterification in SCFs increases the diffusion rates, which results in higher reaction rates. Of all SCFs, SCCO2 is the most noticeable, because it is nontoxic, nonflammable, cheap, easily available, and of mild critical properties (31.1 C and 7.38 MPa). Another reason is that the favorable transport properties of SCCO2 can accelerate mass-transfer-limited in enzyme-catalyzed reactions. Therefore, most researchers in SCFs have used SCCO2 as a solvent. However, it is reported that the biodiesel yields of enzyme-catalyzed synthesis in SCCO2 were between 40% and 90% and the operational stability of the enzyme was then found to be lower (Oliveira and Rosa, 2006). However, compared with the synthesis in supercritical alcohol, synthesis catalyzed by the enzyme in SCCO2 is carried out at near-ambient temperature. Because the products and the enzyme do not dissolve in carbon dioxide at room temperature, separation can easily be achieved by reducing the pressure (Madras et al., 2004).
Mechanism and kinetics of transesterification The overall process of transesterification reaction is usually considered as a sequence of three continuous reversible steps. In the first step, TG are turned into diglycerides (DG). Then DGs are turned into monoglycerides (MG). Finally, MGs are turned into glycerol. During these three steps of transesterification reaction, fatty acid esters are produced. According to the reaction, 3 mol of alcohol is required to react with 1 mol of TG. An excess amount of alcohol is generally required to drive the reversible reaction to the right side
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Table 7
Several representative examples of microbial production of biodiesel
Biodiesel-producing whole cell Engineered gene
Feedstock
Biodiesel yield
In vitro route (intracellular E. coli E. coli E. coli S. cerevisiae P. pastoris A. oryzae A. oryzae
Vegetable oils, methanol Waste grease, methanol Waste grease, methanol Soybean oil, methanol Waste cooking oil, methanol Rapeseed oil, ethanol Soybean oil, methanol
78–100% 97% 87–95% 71% 82% 94% 98%
Olein, methanol Palm oil, methanol Plant oil hydrolysates, methanol
90% 90% 90%
Soybean oil, methanol Soybean oil, methanol Soybean oil, methanol Soybean oil, methanol Olive oil, methanol
78.3% 83.14% 90% 95.4% 89.4%
Soybean oil, methanol Soybean oil, methanol Waste cooking oil, methanol
95% 95% 87%
LB medium, glucose, sodium oleate LB medium, ethanol
1.28 g L 1 1.99-2.01 mg g
lipase) Proteus sp. lipase Serratia marcescens lipase T. lanuginosus lipase, Candida antarctica lipase B R. oryzae lipase T. lanuginosus lipase Fusarium heterosporum lipase Fusarium heterosporum lipase, Aspergillus oryzae lipase (mdlB) A. oryzae A. oryzae lipase (mdlB) A. oryzae Geobacillus thermocatenulatus lipase A. oryzae C. antarctica lipase B In vitro route (surface displayed lipase) S. cerevisiae R. oryzae lipase P. pastoris Rhizomucor miehei lipase P. pastoris R. miehei lipase, C. antarctica lipase B P. pastoris T. lanuginosus lipase, C. antarctica lipase B E. coli Staphylococcus haemolyticus lipase In vitro route (extracellular lipase) P. pastoris R. oryzae lipase P. pastoris R. miehei lipase, Penicillium cyclopium lipase P. pastoris T. lanuginosus lipase In vitro-in vivo hybrid route E. coli Pdc, Adh, WS/DGAT E. coli Plant thioesterases (ucFatB, ccFatB), WS/DGAT E. coli S. cerevisiae S. cerevisiae In vivo route E. coli E. coli E. coli E. coli
E. coli S. cerevisiae S. cerevisiae
Pdc, Adh, WS/DGAT
LB medium, rice straw hydrolysate/restaurant oil wastes, sodium oleate Overexpression of Gcy1,Dak1,Gup1, and WS/DGAT; Galactose, YNB, glycerol, sodium oleate deletion of GPD2 and FPS1 Candida sp. lipase 2 YPD medium, ethanol Overexpression of Pdc, Adh, accBACD, tesA’, WS/ DGAT, fadD; knockout of fadE Overexpression of Pdc, Adh, accBACD, tesA’,WS/ DGAT (double copies), fadD; knockout of fadE Overexpression of Pdc, Adh, TES, ACL, WS/DGAT, xylanases (xyn10B and xsa); knockout of fadE Overexpression of Pdc, Adh, TES, ACL, WS/DGAT, xylanases and cellulase (gly43F and xyn10B, cel3A and cel); knockout of fadE Overexpression of FAT, FAMT, MAT Acc, wax ester synthase (WS) Disruption of DGA1, LRO1, ARE1, ARE2 and POX1; overexpression of wax ester synthase (WS)
1
0.249-1.27 g L 1
520 mg L 1 11.4 mg g 1
Modified LB medium
922 mg L 1
Minimum medium, glycerol
813 mg L 1
Glucose, xylose, hemicellulose
3.5-674 mg L 1
IL-pretreated switchgrass, xylan/cellobiose, glucose 71-405 mg L 1
M9 minimal medium, glucose SD medium, glucose Glucose, YNB
1.87–22 mM 8.19 mg L 1 17.2 mg L 1
Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
so as to obtain more fatty acid esters. The thermal transesterification reaction can also be thought of the following way: TG reacts with methanol to produce DG which reacts with alcohol to generate MG in the following reaction. At last, MG reacts with alcohol to produce glycerol. At each reaction step, 1 mol of alcohol is consumed while 1 mol of fatty acid ester is produced. This kinetic mechanism can be described by Fig. 3. Kinetics of alcoholysis of triacylglycerols for biodiesel production was usually measured using short chain alcohol as acyl acceptor. The kinetics mechanism for transesterification reaction has been proposed to be represented by Ping-Pong Bi Bi model. For the Ping-Pong Bi Bi with alcohol inhibition, the expression of reaction rate was described by the following equation: ½Strigly½Salcohol Vi ¼ Vmax KMtrigly½Salcoholð1 þ ½Salcohol=KIalcoholÞ þ KMalcohol½Strigly þ ½Strigly½Salcohol
(1)
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Fig. 2 Three types of genetically engineered lipases can be used to mediate in vitro biodiesel production: they can be produced intracellularly by the microbial cell, displayed on its surface or secreted in the medium (oval represents microbial cell used for expressing various forms of lipases). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Table 8
Comparison of different engineered microbial whole cells used for biodiesel production
Biodiesel-producing microbe
Expression level
In vitro route (intracellular lipase) E. coli Low S. cerevisiae Moderate P. pastoris High A. oryzae High In vitro route (extracellular lipase) P. pastoris High In vitro route (surface displayed lipase) S. cerevisiae Low P. pastoris Low E. coli Low Ex vivo route E. coli Low S. cerevisiae Low In vivo route E. coli Low S. cerevisiae Low
Cell growth
Cell mass
Immobilization
Substrate transfer
Reaction time
Biodiesel yield
Hours to days Days to one week Days to one week Days to one week
Low Moderate High Moderate
Not required Not required Not required Required
Difficult Difficult Difficult Difficult
Moderate Long Long Long
High Moderate Moderate High
Days to one week
High
Not required
Easy
Short
High
Days to one week Days to one week Hours to days
Moderate High Low
Not required Not required Not required
Easy Easy Easy
Short Short Short
High High High
Hours to days Days to one week
Low Moderate
Not required Not required
Moderate Moderate
Moderate Moderate
Low Low
Hours to days Days to one week
Low Moderate
Not required Not required
Moderate Moderate
Moderate Moderate
Low Low
Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 3
Three steps of reversible transesterification reaction.
where, Vi stands for the initial reaction rate, Vmax stands for the maximum reaction rate, KMtrigly stands for the Michaelis–Menten constant for TGs, and KMalcohol is the Michaelis–Menten constant for alcohol. KIalcohol stands for the inhibition constant for alcohol. Strigly and Salcohol stand for the initial concentration of TGs and alcohol, respectively. The Ping-Pong Bi Bi mechanism could also have been used in the kinetic studies of esterifications of long-chain fatty acids catalyzed by lipases. It can be described as the following steps: (a) as the nucleophile, the oxygen in the OeH group of lipase is added to generate enzyme–substrate complex; (b) first, proton is located in the conjugate acid of the amine, then, it moves to the alkyl oxygen atom of the substrate, resulting in a glycerol moiety. If the initial reaction substrate was TG, then a DG will be generated. Similarly, if the substrate is DG, then MG will be generated; (c) In the methanol molecule, the oxygen atom is added to the acyl enzyme intermediate, which contains the carbon atom of the C]O. Then, the complex, which consists of acylated enzyme–alcohol, will be formed; (d) the enzyme oxygen atom is removed from the complex, and a proton migrates from the conjugate acid of the amine, generating FAME (Al-Zuhair et al., 2007).
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If the alcoholysis of triacylglycerols for biodiesel production is only inhibited by alcohol, the Ping-Pong kinetic model with competitive inhibition can be described by the following equation: V¼
V h max i 1 þ ½KAA 1 þ K½BiB þ ½KBB
(2)
where, V stands for the initial rate of reaction, Vmax stands for the maximum rate of reaction, KA stands for the binding constants of the fatty acid (A) and KB stands for the binding constants of the alcohol (B), and KiB stands for the inhibition constant of the alcohol. The studies of kinetics and the mechanism of enzymatic-catalyzed transesterification reaction can provide the essential parameters for the selection of the best lipase system in biodiesel production. However, the mechanisms of transesterification reaction for biodiesel production by lipase catalysis have not yet been thoroughly studied, thus none of the reported explanations has been widely accepted unambiguously. The complete explanations of the mechanism on the transesterification alcoholysis of triacylglycerols for biodiesel production need further, deeper investigation.
Engineering of Microorganism to Biosynthesize Biodiesel In addition to in vitro lipase mediated biodiesel production, genetic engineering and metabolic engineering of a chassis like E. coli and S. cerevisiae to produce biodiesel based on genetically modifying and system regulation of the fatty acid biosynthetic pathway or/and introducing heterologous or homologous ethanol biosynthetic pathway has been recently emerging (Table 8). The chassis can be metabolically redesigned and systematically regulated to create a balanced biodiesel-producing microbial cell factory (Figs. 4 and 5). Fatty acid biosynthesis and the degradation pathway are well elucidated in model microorganism E. coli. Fatty acyl is a central
Fig. 4 Schematic representation of microbial production of biodiesel via in vitro (A), ex vivo (B and C) and in vivo (D) routes. Depending on each case, the substrates (feedstock) can be provided exogenously or synthetized by the microbial cell. The microbial enzymes required, including lipases and other biodiesel biosynthetic enzymes, are indicated inside the cells (oval shapes). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 5 A dynamic sensor-regulator system containing the repressor gene fadR and FadR-regulated promoters (PmodB and PmodC) for enhancing FAEE titer via bio-sensing intermediate fatty acyl-CoA. Adapted from Zhang et al., 2012.
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metabolite used as a precursor for multiple fuels and chemicals. Fatty acid ethyl ester as a fatty acid-derived product can be de novo biosynthesized by engineering of fatty acid biosynthetic and degradation pathways. Overexpression of key enzymes involved in synthesis steps and knocking out the critical enzymes responsible for fatty acid degradation can accumulate fatty acyl-CoA. Addition of exgenous ethanol or introducing an ethanol formation pathway could confer the host strain fatty acid ethyl ester-producing capacity. Under optimized fed-batch microbial fermentation, an engineered E. coli produced biodiesel with a titer of 922 mg L 1 fatty acid ethyl esters (Duan et al., 2011). Especially, using a genetically tractable oleaginous host such as Y. lipolytica as a chassis can supply a sufficient fatty acid pool, which would significantly increase the biodiesel yield. Currently, we are developing oleaginous Y. lipolytica yeast as a cell factory for biodiesel production according to the route described in Figs. 6 and 7.
Fig. 6 Proposed routes for metabolic engineering of oleaginous Y. lipolytica yeast for biosynthesis of biodiesel based on lipase (dotted line and enzyme in blue represent introduced pathway, enzymes colored in green require overexpression, represents knocked out enzymes, red intermediates represent central metabolites in this engineered pathway). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
Fig. 7 Proposed routes for metabolic engineering of oleaginous Y. lipolytica yeast for biosynthesis of biodiesel based on WS (dotted line and enzyme in blue represent introduced pathway, enzymes colored in green require overexpression, represents knocked out enzymes, red intermediates represent central metabolites in this engineered pathway). Adapted from Yan, J., Yan, Y., Madzak, C. and Han, B. (2015). Harnessing biodiesel-producing microbes: From genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway. Critical Reviews in Biotechnology 1–11.
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Biodiesel Production and Technologies
Prospect and Conclusion Although alkali-catalyzed biodiesel production has already achieved commercialization, in vitro enzymatic conversion of costeffective feedstock to biodiesel based on liquid free lipases, various immobilized lipases, and whole cell lipase biocatalysts would come to be maintream technologies due to their greener and cleaner properties. Significantly, in vivo de novo biosynthesis of biodiesel from glucose, glycerol, and even lignincellulosic biomass derived sugars, represents the frontier of biodiesel production. Especially, the combination of genentic engineering, metabolic engineering, systems biology, and synthetic biology technologies will advance the biodiesel-producing microbial cell factory from laboratory scale to industrial scale in the near future. To make biodiesel production more economical and competitive, low cost oils, including waste cooking oils, high acid value oils, forestry oils, and microbial oils, are primary options. From the perspective of environmentally friendly biodiesel manufacturing, enzymatic transformation of cost-effective oils to biodiesel via a variety of immobilized lipases and whole cell biocatalysts should be seriously considered and thoroughly investigated. Furthermore, redesigning and constructing a biodieselproducing microbial cell factory from lignincellulosic biomass derived sugars is a promising technology that is worth developing.
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