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for bio-diesel production in a sustainable manner. Biocatalytic processes to produce bio-diesel or biofuel is the need of time to reduce the emission of greenhouse gases produced from conventional diesel or fossil fuels. Lipases with excellent biochemical and physiological properties are most commonly used to catalyze the transesterification process for biodiesel production. Lipases obtained from microbes such as bacteria and fungi produce 70%–95% ethanol and methanol. Biodiesel is usually composed of fatty acid alkyl esters which are mono-alkyl esters of either fatty acid methyl esters or fatty acid ethyl esters depending upon the alcohol (acyl acceptor) being used in the reaction. Factors such as bioreactor type, acyl acceptor, temperature, and glycerol can affect the enzymatic transesterification reaction. Recombinant enzymes such as recombinant lipases can be employed to obtain higher percentage of biodiesel due to their high specificity and biocatalytic activity for different substrates used for biodiesel production.

      Keywords: Lipases, biodiesel, biocatalysis, biofuels, Novozyme, free fatty acids, ethyl acceptors

1.1 Introduction and Background

      Biofuels are crucial for the conservation of our natural environment and the climate. Biofuel such as bioethanol can be used for energy generation purposes which are currently being produced by fossil fuels such as petrol, diesel, and kerosene oil [1]. Being non-renewable energy sources, fossil fuels will not only deplete from the planet earth but will also leave a long-term impact on the globe both in terms of economy and climate change. Apart from being limited natural fuel reserves, there are countless reasons available that justify the need of natural and eco-friendly energy sources such as bio-fuels. Transportation, power generation, and house hold appliances use fuels directly or indirectly and for that purpose we are almost dependent on fossil fuels [2]. If efficient and robust methods and technologies are not worked out, we might come to a permanent stand still condition in the future when all our natural fossil fuel reserves will be vanished. The use of fossil fuel produces gases such as carbon dioxide (CO₂), carbon monoxide (CO), sulfur oxides (SO_x), and nitrogen oxides (NO_x) which are unhealthy for human beings causing health issues such as asthma, skin diseases, and even cancers [3]. These by-products of fuel consumption affect not only human but also animals and plants on a broader view. Plant production and growth rates are highly effected by the changing environmental and climatic conditions due to heavy use of fossil fuels and their derivatives such as plastics [4].

      Vehicular CO₂ emission in the past decade was 20%, and it is estimated that by 2030, it will reach up to 80%. Liquide biofuels got prominence with the automobile industry. Peanut oil was used to make biofuel, i.e., biodiesel by Rudolph Diesel in 1898. Henri Ford who was the founder of Ford Company an automobile industry was also convinced by the idea of using biofuels in his automobile. During World War-II, Germany used biomass-based fuels for their machines which is the evidence of its use back in 1940s. The utilization of biofuels was presented, but after two major oil crises, first was in 1973 and second in 1978, and brought back its importance to public again. Biofuels that are produced using a large number of biomass sources are a sustainable solution for the environment and biosphere conservation. Being renewable energy resources and eco-friendly to the environment and life on earth, these are highly desirable products produced from renewable biomass substrates [5].

Currently, biofuels from various agricultural sources such as soybean oil, rapeseed oil, recycled waste oils, and waste plant residues are being studied. Depending on the feedstock type, processing technology and their developmental level, biofuels can be classified into first-, second-, and third-generation biofuels. Biofuels produced directly from edible feedstock such as crops, sugars, and edible oil using conventional techniques are considered as first-generation biofuels [6]. Non-edible feedstock such as waste crop residues like lignocelluloses and waste vegetable oils are required to produce second-generation biofuels which are comparatively economical and more sustainable as there is no food versus fuel competition. Highly advanced methods are used to produce second-generation biofuels which has certainly less flaws and ultimately improved to get greater yield [7]. We are currently in the phase of second-generation biofuels. Most of the processing techniques for second-generation biofuel production are not available at commercial level. One must think that the land dedicated for edible feedstock/crops will be compromised if we start cultivating non-edible crops in that land. Marginal lands can be used for the cultivation of grasses and other plants that are 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,

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