not a food for human or nor a fodder for animals on a larger scale. These plants or marginal grasses can be used for the production of second-generation bio-fuels. There have been a lot of research investigations to produce biodiesel using non-edible plant oils such as keranja oil, Jatropha curcas oil, tobacco oil, Calophyllum inophyllum oil, and castor oil [8]. Jatropha is an effective source of biodiesel production because of 30%–50% oil contents in its seeds [9]. The actual precursors of most of the second-generation biodiesel production are waste oils either in the form of waste cooking or industrial oils or animal fats. The utilization of these waste materials as feed stock helps in managing and disposing of waste material, which is one of the biggest problem for earth, for the benefit of environment [10]. In order to comprehend different biofuels, we can categorize them into four types which include biodiesel, bioalcohol (biomethanol, bioethanol, biobutanol), biogas, and biohydrogen. The most widely used biofuels are liquid biofuels such as biodiesel and bioethanol. Biofuels can be blended with other petro-based fuels in order to manage and enhance quality and quantity of fuel. Biofuel production includes chemical, thermal, and enzymatic methods. Among all methods, the most effective way to produce biofuels is through enzymes or biocatalysts [11]. Enzymes are becoming the focus of research to produce biofuels because of their advantages over other biofuel production techniques [12]. In this chapter, we discuss biodiesel production using biocatalytic processes and methods where different microbial enzymes (obtained from microorganisms) are used.
1.2 Importance of Biodiesel Over Conventional Diesel Fuel
Chemically, biodiesel is composed of fatty acid alkyl esters (FAAEs) which are mono-alkyl esters of either fatty acid methyl esters (FAME) or fatty acid ethyl esters (FAEE) depending upon the alcohol (acyl acceptor) being used in the reaction [10]. Rudolf Diesel, the inventor of diesel engine, first used biodiesel in 1900 but that was highly viscous so that engine could not run effectively for a longer time [13]. Biodiesel is very suitable alternative to diesel fuel because of its remarkable properties and advantages, i.e., biodiesel carries 4.5 times greater energy than fossil fuel [14] and similar in chemical structure and energy content to conventional diesel [15]. It reduces approximately 85% carcinogenic compounds emission that is why it is very less toxic than conventional diesel fuel, free of sulfur, free of polycyclic aromatic hydrocarbons and metals, biodegradable, high cetane number (CN), and flash point [16]. It has the potential to reduce pollutants and emission of greenhouse gases [17] and is 66% more efficient lubricating agent than petro-diesel, which enhances life and performance of engine [18]. Blending of biodiesel with petro-diesel fuel that can affect important properties of fuel such as flash point, CN, kinematic viscosity, and lubricity is enhanced. It also decreases exhaust emissions and heat of combustion [19]. The largest biodiesel producer is EU (European Union) and biodiesel accounts 80% of the overall transport fuel in EU [20–22]. Biodiesel produced from different resources will have different composition and properties, but it must fulfill the standards and requirements of international standards of American society for testing materials and EU standards for biodiesel. Biodiesel has lots of applications such as it can be used as a fuel for aviation purposes [20], for electricity production using generators [21, 22] and in diesel fueled marine engines, because of its nontoxic and biodegradable properties environmental impacts on engines can be reduced. Alcohol type, quality of substrate that is to be converted, catalyst used, temperature of the reaction, and alcohol-to-oil molar ratio determine the performance of biodiesel production [23–25].
1.3 Substrates for Biodiesel Production
Biodiesel feedstock accounts for 60%–80% of the total cost; therefore, appropriate feedstock is required for economically valuable production of biodiesel [26]. In order to obtain economically beneficial and sustainable biodiesel, feedstock must be easily available, cheap, and sustainable. Feedstock is selected on the basis of biodiesel production that must be compatible to chemical composition and properties of feedstock to be used, percentage per dry biomass, agricultural potential, yield per hectare, and geographical region of that feedstock [27]. For example, soybean oil, palm oil, coconut oil, and rapeseed oil are mainly used as feedstock in US, tropical countries like Indonesia, coastal areas, and European countries, respectively. Cultivation and climate conditions of the feedstock production area are also considered for its selection [28]. Depending on the nature, there are two types of feedstock for biodiesel production. First is the lipid raw material and second includes alcohol feedstock. Lipid sources can be divided into three categories, i.e., oils derived from plant sources (edible and non-edible oils), waste oils (waste cooking oils, industrial wastewater, lard, yellow grease, and animal fats), and oils from oleaginous microorganisms such as bacteria, fungi, and microalgae [29]. Properties of biodiesel like cold filter plugging point and oxidation stability are determined from the feedstock used for production. Feedstock properties like moisture content, impurities, content, and composition of free fatty acids (FFAs) affect the performance of engine [27, 28]. Composition of fats and oils including monoglycerides, diglycerides, and triglycerides are used for biodiesel production. Utilization of edible plant oils as feedstock is an expensive way for biodiesel production that leads to imbalance in food market and industry. It is also associated with some environmental problems like disruption of vital soil resources and deforestation due to mass propagation [29]. In order to solve problems linked with edible plant oils, the best alternate is the production of second-generation biodiesel which is produced by using non-edible (inedible) feedstock which are more favorable than edible oils due to reduction in cost and waste pollution, lower aromatic, sulfur contents, and high calorific value [8]. Inedible oils involve inedible plant oils, industrial waste, cooking oils, animal fats, and microalgal oils. Inedible oil producing plants have certain remarkable features that make them favorable to use, for example, they can be managed to grow in arid and semi-arid conditions and they do not require fertilizers and moisture for growth [24].
Repeated use of fried vegetable oils at high temperature leads to the production of waste cooking oils. Moreover, chemical composition of waste cooking oil is totally dependent on the oil from which it is derived. Hydrogenation, oxidation, and polymerization are the main chemical reactions that lead to production of very toxic and detrimental compounds for consumption. Fatty acid content of these oils lies in the range of 0.5% to 15% which is very much higher than refined oil having fatty acid content less than 0.5%. The waste cooking oil is known as yellow grease if the fatty acid content is less than 15% and it is called low value brown grease if the fatty acid content is higher than 15% [28]. Animal waste products like lard, tallow, animal fat, poultry fat, fish oil, and pork fat are also very effective feedstock for biodiesel production [30, 31]. Animal-based bio-diesel is a good lubricating agent and has high percentage of saturated fats which decreases sedimentation risk and low temperature fluidity. Moreover, it increases oxidative stability and cold filter plugging point of biodiesel which are the characteristics of good quality biodiesel. Utilizing these waste materials is an effective solution to encounter waste disposal. Apart from all these mentioned advantages of non-edible or waste oils, there are also some shortcomings or disadvantages, for example, low oil yield, higher carbon residue, unsaturated fatty acid content, and low volatility [29]. In some cases, large plantation land for inedible oils is required compared to edible ones, e.g., Pongamia pinnata and Jatropha has 2–50 folds less oil yield per hectare than palm oil so that is why they require much area to meet the demand [31]. Because of the drawbacks associated with second-generation biodiesel, scientists are looking for more efficient methods for biodiesel production. Biodiesel production using oleaginous microorganisms like bacteria, algae, microalgae, and fungi are considered as the future of biodiesel production that can meet global biodiesel demand for transportation fuels and other energy consuming applications [32]. Microbial oils are better than other plant oils because of their short life cycle and rapid growth, less requirement of space, labor, and easier scaling [33–35]. Microalgae as a feedstock is very effective because of its enormous advantages like they have high oil yield, can grow in salty and waste waters, use of non-arable land, and growth in 24 hours so multiple harvesting in a year is possible. If we give land area for microalgal growth then according to an estimate, only 2% of the US cropping land is enough for meeting 1/3 demand of US transportation fuels and less than 5% land is required to completely replace all transportation fuels [36–38]. Moreover, dry algal biomass can accumulate more than 80% oil without water and they
