Bio-based chemicals from biorefining: lipid and wax conversion and utilization
Y. Yang and B. Hu, University of Minnesota, USA
Abstract:
This chapter reviews current bio-based lipids and wax, the chemical and biological production routes, and their applications. Lipids comprise a variety of naturally occurring compounds, such as fats/oils (triglycerides), phospholipids, diglycerides, monoglycerides, steroids and waxes. Lipids and wax are currently produced from petroleum, as well as plants and animals. Many microorganisms, like bacteria, fungi and microalgae, can also accumulate large amount of lipids and waxes in their cell biomass. Their microbial synthesis is sustainable and the microbial-derived lipids and wax are compatible with the current petroleum-based products. Lipids and wax can be applied as nutraceuticals, pharmaceuticals, fine chemicals and fuels.
Key words
lipids; waxes; microbial production; triglyceride; biodiesel; applications of lipids and waxes
21.1 Introduction
The term ‘lipid’ is very general with various definitions that broadly refer to molecules that can be dissolved in organic solvent instead of water. In this case, lipids can include a wide range of compounds, including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides, phospholipids, and others. Lipids can also be defined as a synonym for fats, which are only a subgroup of general lipids called triglycerides. Wax is an ester of a long-chain alcohol and a fatty acid, a subgroup of the broadly defined lipids. However, wax is usually listed in conjunction with lipids, whereas the term ‘lipids’ as used here only encompasses molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as other sterol-containing metabolites, such as cholesterol. Both terms (‘lipids’ and ‘waxes’) are used in this chapter, which mainly discusses the types and properties of lipids and waxes, their sources of generation, conversion, and utilization. In addition, the current available extraction and analysis methods are discussed. Finally, future research for the lipid and waxes biorefineries is highlighted.
21.2 Types and properties of lipids and waxes
Lipids are a group of chemicals that comprise a variety of naturally occurring compounds. They include a diverse range of compounds such as triglycerides, monoglycerides, diglycerides, phospholipids, terpenoids, carotenoids, and steroids. Lipids mainly serve as energy storage in animals and plants and also as the structural component of the cell membrane. Waxes mainly serve as energy reserves, as well as surface protection. Some lipids are also used as hormones and vitamins for metabolism. Most lipids and waxes are insoluble in water (hydrophobic), but some types of lipids and waxes can be soluble in both water and oil (amphipathic).
21.2.1 Triglycerides
The mostly commonly defined lipids are triglycerides, also called fats, which are the ester of glycerol and three fatty acids. Triglycerides are the major constituents of most natural fats and oils, including vegetable oil and animal fats. A monoglyceride is the monoester of glycerol and one fatty acid; a diglyceride the diester of glycerol and two fatty acids. Triglycerides contain more than twice the energy (38 kJ/g) as carbohydrates and proteins, and they serve as fuel storage in the adipose tissue in humans and seeds of plants. Monoglycerides and diglycerides are intermediates in the degradation of triglycerides. They can be found in cell extracts and may have distinct and important biological properties.
21.2.2 Phospholipids
Phospholipids are mostly made from glycerides by substituting one of the three fatty acids by a phosphate group with some other molecule attached to its end. The other form of phospholipids is sphingomyelin, which is derived from sphingosine instead of glycerol. Phospholipids are soluble in both water and oil (amphiphilic) because the hydrocarbon tails of two fatty acids are still hydrophobic, but the phosphate group end is hydrophilic. Phospholipids are the major component of cell membrane to form lipid bilayers. Figure 21.1 shows the representative structure of common lipids (Gunstone et al., 2007).
21.2.3 Wax esters
In addition to the esters of fatty acids and glycerol, the other type of fatty acid ester is wax esters. Wax esters are long chain esters of fatty acids and alcohols with chain lengths of 12 carbons or more. Wax esters have a variety of functions in organisms, such as surface protection (Gunstone et al., 2007), energy reserves (Lee, Hagen et al., 2006), and constituents of the swimbladder in some myctophid fish (Phleger, 1998). High contents of wax ester can also provide thermal insulation and energy supplies under undesirable conditions (Nevenzel, 1970). Waxes are used as important ingredients in cosmetics, pharmaceuticals, lubricants, plasticizers, and polishes and the other chemical industries (Hallberg, Wang et al., 1999). Figure 21.2 shows the structure of wax ester.
21.2.4 Terpenoids
Terpenoids are hydrocarbon-like compounds produced by many plants, are the major constituents of the essential oils, and are universally present in small amounts in living organisms. They are a diverse class of natural products that have many functions in the plant kingdom and in human health and nutrition (Roberts, 2007). Due to their biosynthesis pathways, the carbon skeletons of terpenoids are oligomers of isoprene; however, many terpenoids undergo further modifications to obtain different structures (Abraham, 2010). Generally, based on the number of the isoprene structure and the length of the carbon chain, terpenoids can be classified as monoterpene (10 carbon), sesquiterpene (15 carbon), diterpene (20 carbon), triterpene (30 carbon), and tetraterpene (40 carbon). Figure 21.3 shows the representative molecule structures for some monoterpenes and sesquiterpenes.
21.2.5 Other lipid compounds
Lipids also contain other organic components that can be dissolved in the organic phase, for example, carotenoids and steroids. Carotenoids are tetraterpenoid organic pigments that are naturally occurring in chloroplasts and chromoplasts. The function of carotenoids includes absorbing blue light for photosynthesis and protecting chlorophyll from photo damage (Armstrong and Hearst, 1996). More than 600 known carotenoids have been discovered and split into two classes: xanthophylls, which contain oxygen; and carotenes, which contain no oxygen. Most carotenoids have 40 carbon atoms and belong to the tetraterpene family. Carotenoids are in the form of a polyene hydrocarbon chain and may or may not have additional oxygen atoms attached. The degree of conjugation and the isomerization state of the backbone polyene chromophore determine the absorption properties of each carotenoid (Cohen, 2011), which is directly linked to their color.
Steroids are known as precursors of certain vitamins and hormones. They are also key constituents in mammalian cell membranes to maintain a proper membrane permeability and fluidity. In animal tissues, cholesterol is by far the most abundant member of steroids. Cholesterol is the principal sterol synthesized by animals and is the precursor molecule for the synthesis of vitamin D and all the steroid hormones. Although cholesterol is important and necessary for the biological processes, high levels of cholesterol in blood is one of the major risk factors for coronary heart disease, heart attack, and stroke.
21.3 Sources of lipids and waxes
Lipids and waxes are currently produced from petroleum as well as plants and animals. All plants contain oils or fats, primarily in their seeds. Lipids and waxes synthesize a huge variety of fatty acids, and triglycerides are the most important form of lipids in plant seeds, followed by the phospholipids. Terpenoids and carotenoids can be found in all parts of the plants, but are relatively concentrated in certain species of plants and microalgae. Fat and steroids are predominantly found in the animal tissue. No significant amount of cholesterol is found in plant sources. Some plants contain phytosterols, which is a cholesterol-like compound and believed to have competition in cholesterol absorption and metabolism (Ostlund, Racette et al., 2003). The biological-derived lipids and waxes can be a good alternative supplement to the current production from petroleum and other fossil fuel origins. However, the limited access to natural lipids and waxes results in a high cost.
21.3.1 Lipids and waxes extracted from plants
The most important oil plants are soybean, palm, rapeseed, and sunflower. During 2011/2012, soybeans accounted for approximately 75% of oilmeal production, while soybean and palm plants account for approximately 60% of plant oil production. Plant oil is also important feedstock for biodiesel production. The most commonly used oil plants for the production of biodiesel are soybean, sunflower, palm, rapeseed, canola, cottonseed, and jatropha.
The primary source of natural waxes produced by plants is the waxy material in the leaf and stem surface. This thin layer of wax has multiple purposes, such as limiting water diffusion and providing protection from insects. Some plants can accumulate a thick coating of wax on leaves (carnauba palm) or have the capability to produce wax ester instead of triglycerides in their seed (jojoba). The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Waxes can also be generated from animal sources, with Beeswax and wool wax as the prime commodities of natural waxes from animal sources (Li, Kong et al., 2010).
One important issue for the use of plant lipids is the limited production of raw material such as soybean oil or vegetable oil, which makes the biodiesel industry suffer in terms of production capacity. Meanwhile, the most costly part of biodiesel production is the cost of feedstock, and current plant oil prices make biodiesel only profitable when it is subsidized. Waste vegetable oils, due to lower prices than original vegetable oils, are considered as a potential low-cost lipid source for biodiesel. In addition to these regular oil plants, a number of wild plant species are capable of producing high amounts of unusual fatty acids, which can be used in high-value industrial and pharmaceutical/nutraceutical applications (Dyer, Stymne et al., 2008).
21.3.2 Lipids extracted from microalgae
Microalgae are generally autotrophic eukaryotic cells that are mostly unicellular with some species that are colonial or filamentous. They can grow autotrophically and/or heterotrophically, with a wide range of tolerance to different temperatures, salinity, pH, and nutrient availabilities. The eukaryotes microalgae referred to include green algae, diatoms, yellow-green algae, golden algae, red algae, brown algae, dinoflagellates, and others; limited species have the capability to accumulate a high content of lipids in their cell biomass (Stoytcheva and Montero, 2011). Some algae have been found to contain more than 80% lipids, which is of great interest for a sustainable feedstock for biodiesel production. Algae oil is being seriously considered because of the large oil yields compared with that for other oilseeds. Research by the National Renewable Energy Laboratory (NREL) showed that 7.5 billion gallons of biodiesel could be produced from approximately 500,000 acres of desert land. Algae farms could also be constructed to use waste streams (either human waste or animal waste from animal farms) as a food source, to extract fertilizer high in nitrogen and phosphorous from algae production, and to provide a means for recycling nutrients. Lipid accumulation occurs within the microalgae cells and varies based on the strain and growth conditions. Table 21.1 lists several promising algae species and their growth performance. Examples include Chlorella vulgaris, which is a commercially important green microalgae due to its high photosynthetic efficiency. Chlorella protothecoides is another single-cell green microalgae, and heterotrophic growth of C. protothecoides supplied with acetate, glucose, or other organic compounds as carbon source, results in high biomass and high content of lipid in cells (Xu et al., 2006).
Table 21.1
Examples of microalgae cultivation for oil accumulation (Heredia-Arroyo, 2011)
| Microalgae | Cultures | Substrates | Growth rate (h) | Lipid content | ||
| AC | MC | HC | ||||
| Chlorella protothecoides | X | X | Glucose, acetate/CO2 | 3.74 g/L in 144 h | 55.2% | |
| Chlorella vulgaris | X | X | X | Glucose, acetate, lactate/CO2 | 0.098 g/h | – |
| Crypthecodinium cohnii | X | Glucose/CO2 | 40 g/L in 60–90 h | 15–30% | ||
| Scenedesmus obliquus | X | X | Glucose/CO2 | Doulbe in 14 h after adaptation | 14–22% | |
| Chlamydomonas reinhardtii | X | X | X | Acetate/CO2 | Exponential during the first 20 h | 21% |
| Micractinium pusillum | X | X | CO2 | 0.94 g/L in 24 h | – | |
| Euglena gracilis | X | CO2 | – | 14–20% | ||
| Schizochytrium sp | Glycerol/CO2 | – | 55% | |||
| Spirulina platensis | X | X | Glucose/CO2 | 0.008/h | – | |
| Botryococcus braunii | X | CO2 | Low growth rate | 20–86% | ||
| Dunaliella salina | X | X | CO2 | – | ∼ 70% | |
AC: autotrophic cultures; MC: mixotrophic cultures; HC: heterotrophic cultures.
Many nutritional and environmental factors control the cell growth and lipid contents. Nutritional factors include organic and inorganic carbon sources, nitrogen sources, and other essential macro- and micronutrients such as magnesium and copper; environmental factors include temperature, pH level, salinity, and dissolved oxygen. Many microalgae species accumulate a higher content of lipids under heterotrophic growth, using organic carbon as its source instead of carbon dioxide and sunlight. Compared with phototrophic algae, the heterotrophic growth process has the advantages of no light limitation, a high degree of process control, higher productivity, and low costs for biomass harvesting (Barclay, Meager et al., 1994). Miao and Wu (2006) reported that the oil content of heterotrophically cultured C. protothecoides was approximately four times greater than that in the corresponding autotrophic culture. Liu et al. (2010) demonstrated that the heterotrophically cultured cells of C. zofingiensis showed 411% and 900% increases in dry cell weight and lipid yield, respectively, compared with increases for autotrophically cultured cells. In addition to lipid production, high value byproducts can be obtained from heterotrophically cultured microalgae, including polyunsaturated fatty acids and carotenoids (Chen and Chen, 2006).
21.3.3 Lipids from yeast and fungi
Besides microalgae, many filamentous fungi species (e.g., Mucor circinelloides or Mortierella isabellina) can also accumulate a considerably high content of lipids (Xia, Zhang et al., 2011; Heredia-Arroyo, Wei et al., 2011). Many oleaginous yeasts were studied for lipid accumulation on different substrates, such as industrial glycerol (Meesters, Huijberts et al., 1996; Papanikolaou and Aggelis, 2002), sewage sludge (Angerbauer, Siebenhofer et al., 2008), whey permeate (Ykema, Verbree et al., 1988; Akhtar, Gray et al., 1998), sugar cane molasses (Alvarez, Rodriguez et al., 1992), and rice straw hydrolysate (Huang, Zong et al., 2009). Dey et al. (2011) screened two endophytic oleaginous fungi Colletotrichum sp. and Alternaria sp. with lipid content 30% and 58%, respectively. Furthermore, the fatty acid profile of microbial oils is quite similar to that of conventional vegetable oils, which suggests oleaginous filamentous fungi as a favorable feedstock for sustainable biodiesel industry (Peng and Chen, 2008; Zhao, Hu et al., 2011). Table 21.2 shows the oil content of common fungi and yeast species (Meng, Yang et al., 2009).
Table 21.2
Oil content of sample fungi and yeast species (Meng, Yang et al., 2009)
| Microorganisms | Oil content (% dry wt) |
Microorganism | Oil content (% dry wt) |
| Yeast | Fungi | ||
| Candida curvata | 58 | Aspergillus oryzae | 57 |
| Cryptococcus albidus | 65 | Mortierella isabellina | 86 |
| Lipomyces starkeyi | 64 | Humicola lanuginosa | 75 |
| Rhodotorula glutinis | 72 | Mortierella vinacea | 66 |
21.3.4 Waxes from bacteria
Fossil waxes are primarily generated by chemical syntheses via Fischer–Tropsch process and olefin (ethylene, propylene) polymerization. Natural waxes are mostly extracted from animals, vegetables, and minerals, and many waxes, such as jojoba oil and sperm whale oil, have important industrial use due to their excellent wetting behavior at interfaces (Hadzir, Basri et al., 2001). However, restricted by their high price and limitation in access, industrial syntheses of wax ester by the esterification reaction of carboxylic acids with alcohols are also applied via chemical (Aracil, Martinez et al., 1992) and enzymatic reactions (Trani, Ergan et al., 1991).
Wax esters were found to be accumulated in some group of prokaryotes, such as bacteria (Ishige, Tani et al., 2003). These microbial waxes are favorable because their composition can be manipulated by the growth condition. Some members of the genus Acinetobacter can store significant amounts of wax ester in cells, which can account for up to 15–25% of the cell dry matter (Kalscheuer and Steinbuchel, 2003). Acinetobacter can grow and synthesize wax esters on a variety of substrates, such as alkanes and aromatic hydrocarbons, as well as acetate, sugars, and acids (Waltermann, Stoveken et al., 2007). In addition, the accumulation of wax esters has also been reported in some species of the genera Fundibacter (Bredemeier, Hulsch et al., 2003), Micrococcus (Russell and Volkman, 1980), and Moraxella (Bryn, Jantzen et al., 1977). The intracellular accumulation of wax esters was found in a few species of prokaryotes, and jojoba plant (Simmondsia chinensis) is the only source of eukaryotes that accumulates wax esters as intracellular storage lipid (Waltermann and Steinbuchel, 2005). Engineering bacteria with wax ester synthase has been studied for the biosynthesis of wax esters for higher production (Kalscheuer and Steinbuchel, 2003), which may substitute current wax production from crude oil or plant (jojoba) and animal oils.
In addition to plants and pure cultures of microorganisms, other sources of lipids and waxes are available. Activated sludge was discovered to have high lipid and wax content because of its high microbial biomass content, and it is technically possible to produce biofuel or biodiesel from activated sludge. A recent study found that more than 25 wt% crude lipid and waxes can be obtained from activated sludge in a food processing company in Taiwan (Huynh, Do et al., 2011). Many other waste materials can also be utilized for the production of lipids and waxes, such as lanolin extracted and purified from the wool wax of wool scour wastes (Lopez-Mesas, Christoe et al., 2005), and sugarcane waxes extracted from rum factory wastes (Nuissier, Bourgeois et al., 2002).
21.3.5 Lipids and waxes generated from lignocellulosic materials and other sources
Due to the limited supply of plant-based resources, lipids and waxes generated via microbial accumulation are attracting more attention based on their availability, relatively low cost, and the development of conversion technology. The theoretical yield for converting sugars to long-chain fatty acids (lipids and waxes) is 32%; most oleaginous cell cultures can reach the yield of 20–25% because some sugars have to be diverted to support the cell growth and metabolism. Therefore, the use of non-starch biomass is critical so that lignocelluloses can be used for organic carbon supply without concerns of using food crops for fuel sources. Lignocellulosic materials are mainly composed of cellulose, hemicellulose, and lignin, whereas only cellulose and hemicellulose can be converted to fermentable sugars for microbial lipid production. Recent studies confirmed conversion of hemicellulose hydrolysate into lipids by oleaginous yeast strains and their tolerance degrees to lignocellulose degradation compounds (Chen, Li et al., 2009; Hu, Zhao et al., 2009; Huang, Zong et al., 2009).
The oleaginous cell cultivation on lignocelluloses shares many similarities with current lignocellulosic ethanol production, whereas cell fermentation is followed by pretreatment and saccharification to release monomeric sugars for microbial utilization (Wang, Wang et al., 2011). Pretreatment refers to the disruption of the naturally resistant structure of lignocellulosic biomass to make its cellulose and hemicellulose susceptible to enzymatic hydrolysis, the end goal being to generate fermentable sugars. Many pretreatment processes, including chemical, physical, physicochemical, and biological pretreatment, have been greatly developed in recent decades (Wyman, Dale et al., 2005). Sugars and phenolic compounds from cellulose, hemicellulose, and lignin, as well as their corresponding degradation products, as 5-hydroxymethyl-2-furaldehyde (HMF), 2-furaldehyde, and acetic acid, respectively, constitute the pretreatment hydrolysate. The types and concentrations of the compounds in the pretreatment hydrolysate depend on the pretreatment technology employed, specific conditions, and varieties of feedstock.
The cellulose hydrolysis/saccharification step for lignocellulosic ethanol fermentation combines enzymatic cellulose hydrolysis with yeast fermentation in a process called ‘simultaneous saccharification fermentation’ (SSF). SSF integrates the whole process and minimizes the substrate inhibition. It also creates some difficulties and challenges due to the higher working temperature of cellulase, compared with the lower temperatures at which most yeast strains grow. Both C5 and C6 monomeric sugars become available after hydrolysis, as well as numerous byproducts that may have inhibitive effects on cell growth, such as acetate, formic acid, furans, vanillin, levulinic acid, furfural, hydroxymethylfurfural, and hydroxybenzaldehyde (Venkatesh, 1997; Aden, Ruth et al., 2002; Yang and Wyman, 2004). Compared with lignocellulosic ethanol fermentation, microbial lipid production has its own distinctions. Many ethanol-producing yeast strains cannot utilize C5 sugar, and are severely inhibited by acetic acid and other byproducts from lignocellulosic hydrolysis, whereas oleaginous fungal strains have a wider range of utilization for both C5 and C6 sugars, less cell inhibitions, and fewer requirements for the hydrolysis.
21.4 Methods to extract and analyze lipids and waxes
The aim of all extraction procedures is to separate lipids and waxes from the other constituents. With the purpose to extract oil, different strategies have been developed. Considering the structure texture, sensitivities, and lipid and wax contents of plant and animal tissues and microbial cells, the methods show great diversity. The most widely used methods include solvent extraction, pressing, carbon dioxide supercritical extraction, and microwave-assisted extraction.
21.4.1 Solvent extraction
Lipids and waxes exist in tissues in many different physical forms. The simple lipids are often aggregates in storage tissues, while complex lipids are usually constituents of membranes and are closely associated with proteins and polysaccharides. To extract lipids and waxes from tissues, solvents must be used that not only readily dissolve the lipids but also overcome the interactions between the lipids and the tissue matrix. In addition, simultaneously perturbing both the hydrophobic and polar interactions is essential (Christie, 1993). The greatest improvement in lipid and waxes extraction was made in 1957 when Folch et al. (1957) described their classic extraction procedure. This procedure remains the most commonly used by researchers around the world and has become the standard against which other methods are judged. A key property of this solvent is the capacity of chloroform to associate with water molecules. With the ratio of chloroform-methanol to tissue greater than 17:1, the equivalent of 5.5% water can be solvated and remain in a single phase (Christie, 1993). At the laboratory scale, the extraction efficiency can reach approximately 90%.
21.4.2 Pressing extraction
Pressing may be the oldest method for oil extraction. In India, approximately 90% of the total 24 million tons of produced oilseeds are crushed using this method (Singh and Bargale, 2000). Pressing is the process of mechanically pressing liquid out of liquid-containing solids. The equipment is simple and sturdy, as well as easy to operate, with short training time. Moreover, this process can be adapted quickly for processing of different kinds of oilseeds and yields a chemical-free protein-rich cake (Pradhan, Mishra et al., 2011). It is claimed that the pressing process can typically recover 86–92% of oil from oilseeds (Singh and Bargale, 2000), while the efficiency is highly dependent on the raw material used in pressing. When using hazelnut seeds to extract oil, the pressing process efficiency can reach the low limit of 6.1% (Uquiche, Jerez et al., 2008). Adjusting pressing parameters, such as an increase of the internal pressure, can improve oil recovery and results in a decrease of the residual oil in the cake (Jacobsen and Backer, 1986). Suitable pretreatment of the oilseeds, such as cleaning, conditioning, decorticating, cracking, flaking, cooking, extruding, and drying to optimal moisture content, will also increase the oil recovery (Zheng, Wiesenborn et al., 2003).
21.4.3 Carbon dioxide supercritical extraction
Carbon dioxide supercritical extraction is one application of the supercritical fluid extraction (SFE) process. SFE is a separation technology that uses supercritical fluid solvent for extraction. Carbon dioxide is the most commonly used supercritical fluid, with other choices including ethanol. Compared with traditional soxhlet extraction, SFE uses supercritical fluid to provide a broad range of useful properties. It eliminates the use of organic solvents, which reduces the problems of their storage, disposal, and environmental concerns. In the extraction process, diffusion coefficients of lipids and waxes in supercritical fluids are much higher than in liquids, therefore extraction can occur more quickly. In addition, no surface tension is present in supercritical fluids, and viscosities are much lower than in liquids, which help the supercritical fluids be able to penetrate into small pores that are inaccessible to liquid. Research has shown that the lipid extraction has the ability to reach more than 90% of theoretical value in a short time (King, 2002). Currently, carbon dioxide supercritical extraction remains at the stage of laboratory research.
Some parameters are generally considered to influence the extraction yields with carbon dioxide supercritical extraction. Temperature in supercritical conditions has no determining influence on extraction yield, while increasing of pressure will increase the extraction rate by increasing the oil solubility (Salgin, Doker et al., 2006; Boutin and Badens, 2009). Moreover, many studies indicate that extraction yield increases with pressure (from 3.63 to 18.63 g CO2 kg− 1 from 20 to 60 MPa) due to the solubility increase of the different compounds (mainly triglycerides) with pressure (Salgin, Doker et al., 2006). Increased moisture content will reduce the extraction efficiency (King, 2002), while the moisture content itself may be affected during the extraction process (Dunford and Temelli, 1997). Pretreatment of the sample before extraction will improve mass transfer by increasing the exchange surface and seed destructuring (del Valle, Germain et al., 2006).
21.4.4 Microwave-assisted extraction
Microwave use has only recently been applied to extraction of plant materials. Unlike conventional heating, which depends on the conduction–convection phenomenon (Mandal et al., 2007), microwave energy delivered directly to materials through molecular interaction with the electromagnetic field generates heat through the sample volume to achieve uniform heating (Venkatesh and Raghavan, 2004). In this manner, the use of microwave-assisted extraction often helps to reduce processing time and save energy during the process (Uquiche, Jerez et al., 2008). When combined with other extraction methods, the microwave radiation works as a pretreatment step and can highly increase the efficiency of pressing extraction from 6.1% to 45.3% (Uquiche, Jerez et al., 2008). Similar results were obtained from other research in which microwave pretreatment contributed a significantly higher extraction yield than carbon dioxide supercritical extraction (Dejoye, Vian et al., 2011). Under a given radiation frequency, major factors that influence dielectric properties include temperature and moisture content, chemical composition and the physical structure of the raw material, and sample density (Venkatesh and Raghavan, 2004).
The most current extraction methods in industry are solvent extraction and pressing, which can achieve decent efficiency and maintain a low cost. Carbon dioxide supercritical extraction can achieve higher efficiency, although the high cost prohibits the industrial application at this stage. The microwave pretreatment of raw material for extraction can improve efficiency, but it is currently difficult to treat large amounts of raw material.
21.4.5 Methods to analyze lipids and waxes
Traditional analysis of lipids and waxes focused on the nutritional aspect. Lipids are one of the major constituents of foods, providing an energy source and essential lipid nutrients. Waxes are also utilized for food purposes. Meanwhile, overconsumption of certain lipid components such as cholesterol and saturated fats can be detrimental to human health. In terms of the food science aspect, some of the most important properties of a food analyte are:
• total lipid and waxes concentration;
• type of lipids and waxes present;
• physicochemical properties of lipids and waxes, such as crystallization, melting point, smoke point, rheology, density, and color;
• structural organization of lipids and waxes (Chauhan and Varma, 2009).
The physicochemical characteristics of lipids and waxes, including solubility in organic solvents, immiscibility with water, physical characteristics, and spectroscopic properties, are used to distinguish these elements from other components in food. Analytical methods are based on these principle characteristics and can be categorized into three groups: solvent extraction, non-solvent extraction, and instrumental methods. For solvent extraction, the methods used are similar to the extraction methods described previously. However, some foods contain lipids that are complexed with proteins or polysaccharides and thus need an extra step to break the bonds to release lipid into easily extractable form (e.g., acid hydrolysis). For non-solvent extraction, the separation of lipid and waxes relies on other chemicals such as sulfuric acid and isoamyl alcohol. Standard methods include the Babcock method, the Gerber method, and the detergent method. In terms of the instrumental methods, a wide variety can be chosen. Major principles include measurement of bulk physical properties such as electrical conductivity, measurement of adsorption of radiation such as nuclear magnetic resonance (NMR) spectroscopy, and measurement of scattering of radiation like ultrasonic scattering.
As the extensive research on biofuel and biodiesel has developed recently, lipids are extracted from various sources and converted into biodiesel. The requirements of lipid analysis are then focused on the identification of components in lipids and the generated biodiesel. For this analysis purpose, gas chromatography (GC) and high-performance liquid chromatography (HPLC) are primary choices. For wax ester analysis, GC, GC-mass spectrometry (GC-MS), and HPLC are also commonly used.
GC is widely used for analysis of volatile compounds. During GC analysis, the sample is evaporated in an injection port and then transported through a column by carrier gas stream. During the transport, compounds are separated by the different retention times required to pass through the column. Detectors are connected at the end of column to quantify the amount of each compound that passes through, and normally a flame ionization detector (FID) is used for hydrocarbons and essential oils (Marriott, Shellie et al., 2001). A variety of GC columns are available to allow highly sophisticated application of samples together within a designed temperature program. When the sample contains unknown components, analysis of these hydrocarbons is often achieved by a connection with GC-MS (Koga and Morii, 2006).
Many compounds are simply not volatile enough to qualify for GC analysis; also some nonpolar compounds such as polyyne hydrocarbons and highly unsaturated fatty acids are too unstable for GC analyses (Abraham, 2010). These components are generally separated by HPLC currently. Similar to GC, the HPLC analysis also requires a column for separation and a detector for quantification. HPLC with ultraviolet or photodiode array detection is most often used (Su, Rowley et al., 2002). Similar to the GC-MS system, a mass spectrometer can also be connected with an HPLC system for the identification of unknown hydrocarbons.
21.5 Utilization of lipids and waxes
Lipids and waxes are mostly used as nutraceuticals, pharmaceuticals, fine chemicals, and fuels. Currently, one of the most attractive utilizations for lipids is to generate biodiesel, which is considered as a replacement for current diesel fuel.
21.5.1 Biofuels
As a liquid form of renewable fuel, biofuels attract great effort from research based on their capacity for application as transportation fuels. Biodiesel is an important approach for alternative biofuels. The most common type of biodiesel is esters of fatty acid, obtained by transesterification of lipid with methanol or ethanol. Biodiesel can be used in pure form (B100) or may be blended with fossil diesel at any rate. The most commonly used biodiesel is B99 because 1% of fossil fuel is applied to inhibit mold growth. Biodiesel is approximately 5% to 8% less efficient than conventional fossil diesel but is compatible with the current diesel engine and can be a total or partial (in the cold regions) replacement for the fossil diesel.
The environmental benefits to replacing fossil diesel with biodiesel are numerous because combustion of biodiesel emits far less pollutants than fossil diesel (except the NOx emission). Also, the entire process is close to carbon-neutral considering that the plant oil used to produce biodiesel is synthesized in agriculture from CO2. The production and utilization of biodiesel are significant in many aspects, for example, increasing the oilseed crop market, providing domestic job opportunities to the rural community, and decreasing the dependence on imported oil; therefore biodiesel has been commercialized around the globe.
21.5.2 Triglycerides
The high viscosity, low volatility, and polyunsaturated character of triglycerides prohibit their direct use in an engine system as liquid biofuel. The most common industrially applied method to convert lipids to biofuel is through the transesterification reaction to convert triglycerides to fatty acid methyl ester (FAME), in which long-chain fatty acids are exchanged from triglycerides by methanol, generating FAME and glycerol (Fig. 21.4). This reaction can also be carried out by ethanol to produce fatty acid ethyl ester (FAEE) instead.
In addition to transesterification, several strategies can change the properties of the lipids to serve as biofuel. The hydrocarbon type of diesel, developed recently with direct decarboxylation of fatty acid or lipid (Fig. 21.5), has been shown to be superior to biodiesel in many aspects due to its resistance to water contamination; for example:
1. biodiesel is very corrosive and therefore it is not suitable for pipeline transportation;
2. biodiesel production requires oils of high quality, and algal oil has difficulty in reaching those requirements; and
3. biodiesel has shorter shelf life, and the technology to deal with biodiesel is far more immature than that for diesel.
Pyrolysis, a method of conversion of one substance into another with the aid of a catalyst in the absence of air or oxygen, is proven to produce non-oxygenated, liquid hydrocarbon mixtures from triglycerides and can be used a diesel fuel additive (Maher and Bressler, 2007).
Microemulsion, defined as clear, thermodynamically stable, isotropic liquid mixtures of oil, water, and surfactant, also shows the capability to produce a mixture of diesel fuel and plant oil with same engine performance (Singh and Singh, 2010). Other research activities in this field include hydrocarbon production from natural oils and fats over homogeneous catalysts; gas-phase selective heterogeneous catalytic decarboxylation of carboxylic acids, such as octanoic, benzoic, and salicylic acid, with palladium and nickel catalysts; unsaturated linear hydrocarbons production from saturated fatty acid and fatty acid esters with a nickel type catalyst; and unsaturated diesel-like hydrocarbons production over palladium catalysts on carbon (Snåre, Kubi ková et al., 2008). The decarboxylation process occurs at high temperature with the help of the catalyst, and many byproducts of plant or algal oils show strong toxicity to the catalyst. The chemical decarboxylation process to produce diesel still needs further development compared with the transesterification process for biodiesel production.
Additionally, the thermochemical biorefinery is an alternative method to produce lipids and waxes directly from lignocellulosic materials. Compared with the traditional lipid and wax production process of extraction and microbial accumulation, the thermochemical biorefinery is a new strategy to convert plant-based materials to chemical and energy materials. This process generates syngas and heat for energy use, with some liquid portion containing lipids and wax components. The process is mainly carried out by pyrolysis and gasification, and the feedstock can come from a variety of sources, such as wood products, agricultural residue, and municipal solid wastes. This biorefinery process includes mainly combustion, gasification, and pyrolysis, while product composition is closely related to the reaction temperature and reaction conditions. In addition to being gas generated, lipids and waxes are primarily liquid product components. This biorefinery process will be more economical and efficient when integrated with industries dealing with large amounts of biomass, such as pulp and paper mill, agricultural residue, and municipal solid waste treatment. Currently, the international corporation TRI (ThermoChem Recovery International, Inc.) has commercialized the thermochemical biorefinery integrated with the pulp and paper industry for production of liquid transportation biofuels, bio-based chemicals, substitutes for petroleum-based feedstock and products, and biomass-based heat.
21.5.3 Terpenoids
In addition to the biodiesel and fatty acid derived hydrocarbons, hydrocarbon-like terpenoids are other important molecules proposed for biofuel. Many terpenoid compounds are natural antibiotics, and they are under investigation for antibacterial, antineoplastic, and other pharmaceutical applications. The concept of terpenoid as biofuels is still new and not widely accepted in the scientific community. However, visionary scientific leaders such as Jay Keasling at the University of California Berkeley have promoted the concept. Many terpenoid molecules contain no oxygen, have a similar structure to hydrocarbon, and have a low boiling point to be easily separated, which makes them capable of directly serving as fuels after being purified through the refinery process.
Currently, the microbial terpenoid production systems have several major limitations to being applied for fuel production. The yield with the microbial production systems is too low (the highest yield for the amorphadiene production, including both headspace and liquid content is 22.6 and 112.2 mg/L, respectively), which is far too low for fuel production at the current stage (Martin, Pitera et al., 2003). Furthermore, the production of terpenoids with microbial systems requires the input of glucose or sucrose, which translates into high cost, lower energy conversion efficiency, and an unsustainable production system. Currently, the production of terpenoids is more suitable as high value drugs for the pharmaceutical industry.
21.5.4 Pharmaceuticals and nutraceuticals
Lipids and waxes are historically applied as pharmaceuticals and nutraceuticals due to their significant role in human metabolism, especially because their many components cannot be synthesized by the human body and thus need to be supplied by intake from external sources. Lipids and waxes applied in these fields are generally considered as high-value products. Their applications have already been mostly driven by the market and are currently well commercialized. The following discussion lists some applications of lipids and waxes in these areas.
Polyunsaturated fatty acids (PUFAs)
PUFAs are fatty acids that contain more than one double bond in their backbone. Figure 21.6 shows the structure of n-3 PUFAs (Mozaffarian and Wu, 2011). The most important PUFAs are omega-3 and omega-6. Omega-3 has the first double bond at carbon number 3, counting from the methyl end, and omega-6 has the first double bond at carbon number 6, counting from the methyl end. Major omega-3 PUFAs in the diet include alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), while major omega-6 PUFAs in the diet include linoleic acid (LA), γ-linolenic acid (GLA) and arachidonic acid (AA). The highest concentrations of the omega-3 PUFAs are found in coldwater fish such as salmon, sardines, and tuna, and omega-6 PUFAs are usually found in plants such as olive oil, sunflower seeds, and flaxseeds.
Omega-3 and omega-6 PUFAs are important structural components of the phospholipid cell membranes of the tissues, which have multiple physiological functions (Simopoulos, 1999, 2002). Omega-6 PUFAs are integral components of skin lipids; AA and DHA are the major PUFAs in the brain, nervous tissue, and retina; and DHA is essential for the visual process in the retina and for proper brain functioning. Since enzymes in the human body are not effective in the generation of precursor lipids to these complex PUFAs, directly increasing the intake of EPA, DHA, GLA, and DGLA has the highest clinical benefits.
Monoglycerides
Monoglycerides are fatty acid monoesters of glycerol and exist in two isomeric forms (Fig. 21.7). Their concentrations are very low in cell extracts, but they are intermediates in the degradation of triacylglycerols or diacylglycerols, and thus can be produced through a chemical process. Monoglycerides are widely used as anti-staling agents and account for approximately one third of the emulsifiers used in the baking industry. Another important use of unsaturated monoglycerides is in drug delivery systems because of their ease of processing and unique properties. When in contact with aqueous media, unsaturated monoglycerides result in a highly viscous cubic phase and provide sustained release for drugs (Chang and Bodmeier, 1997).
Carotenoids and cholesterol
Carotenoids constitute one of the most widespread classes of yellow, orange, and red natural pigments found in bacteria, fungi, and photosynthetic organisms, and also in eukaryotes, estimated to synthesize 108 tons every year (Cohen, 2011). Humans and animals are mostly incapable of synthesizing carotenoids; instead they obtain carotenoids from their diet for most antioxidant activity. Therefore, carotenoids are substantially hydrophobic antioxidants, widely utilized in medicine as antioxidants or in food chemistry as colorants. Similar to carotenoids, cholesterol, as the most abundant member of steroids, is an important component in the hormonal system. Cholesterol is a building block for cell membranes and for hormones such as estrogen and testosterone. The liver produces approximately 80% of the body’s cholesterol endogenously, while the remainder is from dietary sources. The major intake of dietary cholesterol is from meat sources including egg yolk, pork, beef, poultry, fish, and shrimp, while no significant amounts of cholesterol are found in plant sources. Humans need a certain level of cholesterol for proper function, but excess cholesterol can cause health problems such as the increased risk of coronary heart disease. Therefore cholesterol is one of the metabolites that people need to monitor and thus has a very limited role in serving as pharmaceuticals or nutraceuticals.
21.5.5 Other applications
Cosmetics and personal care products
Many lipid and wax products are used for cosmetics and personal care products.
• Essential oils – a wide category of hydrophobic liquid with volatile aroma compounds extracted from plants – are used in perfumes, cosmetics, soaps, and other products such as food and drink flavoring and household cleaning products. Among all lipid products, waxes are especially widely applied in the cosmetic industry, mainly applied as a coating agent for their excellent wetting behavior at interfaces.
• Beeswax – a natural wax produced in the honeybee hive – is widely used for skincare product and food additives.
• Lanolin – a wax ester extracted from wool scour wastes – is used mainly for cosmetics, pharmaceutical formulations, and baby care products (Lopez-Mesas, Christoe et al., 2005).
• Octacosanol – a straight-chain 28-carbon primary fatty alcohol that exists in plants' epicuticular waxes and rice bran wax – is used to improve exercise performance including strength, stamina, and reaction time (Chen, Cai et al., 2005).
The list is long of similar products that use lipids and waxes as main ingredients, which are well known to the public.
Lubricants
Lubricants, a substance (often a liquid) introduced between two moving surfaces to reduce friction, are traditionally generated from petroleum-based oil. In light of the excess amounts of lubricants that are lost into the environment, biodegradable lubricants have been considered as a suitable replacement for the petroleum-based ones. Compared to the lubricants made of petroleum, vegetable-based lubricants are much more biodegradable but inferior in many other technical characteristics. Current biodegradable lubricants account for only 2% of global consumption of lubricants; however, the market for biodegradable lubricants is expected to grow 5–10% annually (Erhan and Asadauskas, 2000). Most laws requiring the use of lubricants that are biodegradable are currently in place in several western European countries, such as Germany, Austria, and Switzerland. The increase of environmental regulations, set forth by governments, will be one of the driving factors that increase the demand for biodegradable lubricants. Meanwhile, research and development has been carried out to formulate biodegradable lubricants with technical characteristics superior or at least compatible to those based on mineral oil (petroleum).
Lubricants generally contain two major components: base stock oil and additives. Base stocks usually comprise more than 80% lubricant, determining the key performance properties of the lubricants (Erhan and Asadauskas, 2000). Natural plant-based oils are the predominant choice for the base stocks of the biodegradable lubricant, especially soybean oil, because it has excellent features such as low volatility, excellent lubricity, favorable viscosity temperature characteristics, and high solubilizing capacity for contaminants and additives. However, vegetable-based lubricants are very limited in many features, preventing them from wider applications. The most serious problem is their poor oxidative and hydrolytic stability. The oxidative instability is primarily caused by the polyunsaturation of the vegetable oil (Erhan and Asadauskas, 2000). Vegetable oils also show poor corrosion protection and the presence of ester functionality renders these oils susceptible to hydrolytic breakdown. Some of these problems can be resolved by avoiding or modifying polyunsaturation in TAG structures of vegetable oils. Another way to improve these properties of vegetable oils is chemical modification of fatty acid chains of triglycerides at sites of double bond and carboxyl groups. In particular, a high degree of branching can lower viscosity index, whereas high linearity can lead to high viscosity index, and relatively poor low-temperature characteristics. In contrast, low saturation can limit oxidation stability, whereas high saturation can result in outstanding oxidation stability (Li, Kong et al., 2010).
Bioplastics
Plastic materials are usually synthetic and most commonly derived from petroleum. Due to the short time of their presence in nature, no enzyme structures are capable of degrading petroleum-based plastics. They may persist for hundreds or even thousands of years, and thus environmental concerns are driving the research into biodegradable plastics. Natural biodegradable plastics are based primarily on renewable resources and can be either naturally produced or synthesized from renewable resources. The major sources of biodegradable plastics include polysaccharides, proteins, lipids, polyesters produced by plant or microorganisms, polyesters derived from bioderived monomers, and miscellaneous polymers, for example (Nampoothiri, Nair et al., 2010).
As the main components in plant oil, triglycerides provide glycerol and a mixture of fatty acids by hydrolysis, and both can be used as building blocks for the synthesis of designed monomers (Warwel, Bruse et al., 2001; Jerome, Pouilloux et al., 2008). Research developed by different groups reveals a growing interest in the reactivity of their double bonds towards olefin metathesis, which enables the straightforward synthesis of a wide variety of monomers (de Espinosa and Meier, 2011). Even though with great potential, plants oil and animal fats are generally not the primary and direct raw materials to generate bioplastics. However, from a biorefinery point of view, bioplastics manufacturing from proteins can be integrated with oilseed processing to maximize the usage of the raw materials. A typical example is the bioplastics generation from cottonseed protein, which can be extracted from the residue after organic solvent extraction for oil production. Cottonseed protein can be modified via crosslinking treatment to generate cottonseed protein bioplastics (CBPs) (Yue, Cui et al., 2012). In this scenario, the cottonseed processing will not only generate oil for biofuel generation, but also CBPs to replace plastics generated from petroleum. Similar concepts can be applied to other types of oilseed processings.
21.6 Conclusion and future trends
Even though lipids and waxes have been explored as fine chemicals for decades, due to the recent energy crisis, generation of liquid biofuel from lipids and wax esters has attracted attention from both academia and industry, it becoming the mainstream of research in the last few years. Numerous projects have been funded by the US Department of Energy (DOE) and the US Department of Agriculture (USDA), as well as the National Science Foundation to study the whole process for generating liquid biofuel products from non-food lipids and waxes. These projects cover a wide range of topics, including feedstock development, biological and chemical conversion technologies, harvesting and logistics, and life-cycle assessment. For example, projects funded from the Biomass Research and Development Initiative (BRDI) program under USDA and DOE are specifically focusing on this area in recent years. The DOE also funded research projects to explore possibilities for other types of drop-in biofuels, for example, hydrocarbons and terpenoids. A typical example is the project led by Professor Larry Wackett at the University of Minnesota and supported by the DOE Advanced Research Project Agency-Energy (ARPA-E) program with $2.2 million to use photosynthetic bacteria that can convert light and carbon dioxide to ‘feed’ a hydrocarbon-producing Shewanella bacteria for scaled-up production (Wackett, Gralnick et al., 2010–2012).
In general, the future of the advanced biofuel production system must meet the following requirements:
1. For agriculture residues usage, especially from the average farmer, the processing facility must be compatible with the distributed biomass production system.
2. The conversion facility must be easy to operate and have a relatively simple operation if a small-size facility is chosen.
3. The conversion process must be adaptable to multiple biomass feedstock.
4. Costs related to harvest of cells and conversion of cell biomass to biofuel must be low.
Extracting lipids from cell biomass and converting them to biodiesel is not an ideal route, due to the complexity of the cell harvesting and oil extraction; however, combining the biorefinery concept to convert the biomass residue after oil extraction as animal feed may bring some extra value to offset the process cost. Direct thermal treatment of cell biomass, for example, pyrolysis, is more robust than biological conversion combined with transesterification reaction; however, the final product of the pyrolysis cannot meet the quality standard of liquid fuel, which therefore significantly affects the technical feasibility of this type of conversion. Although no process has been proven to fit all these requirements while still reaching commercial feasibility, further scientific research will advance these processes to be closer to industrial reality.