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1 Introduction

Biodiesel is a renewable and sustainable alternative to the petroleum fossil fuels. Biodiesel can be produced from variety of feedstocks such as edible, non-edible oils, animal fats, and microalgal lipids (Ma and Hanna 1999; Sharma et al. 2008). Fast growth rates and high lipid accumulation have proven microalgae as a promising feedstock for biodiesel production (Chisti 2007; Rawat et al. 2013). Catalytic conversion of feedstock oil to fatty acid alkyl esters (FAAE), i.e., biodiesel is the most widely used method (Helwani et al. 2009). The conversion process depends upon number of factors such as quality of feedstock oil, choice of catalyst, acyl acceptor, use of solvent, and reaction parameters. The recent advances such as process intensification by microwave and ultrasonication have improved the yields of catalytic conversion of lipids to biodiesel. This chapter deals with the brief overview, recent advances, and challenges in catalytic conversion of microalgal lipids to biodiesel.

2 Microalgal Lipids as a Feedstock for Biodiesel

Microalgal lipid is considered as a greener and sustainable feedstock for biodiesel synthesis. Microalgae have faster growth rates and higher lipid accumulation capability than the terrestrial plants. Microalgae also offer other environmental benefits such as CO2 mitigation and wastewater utilization. Microalgae have shown lipid accumulate of up to 20–70% lipid per dry cell weight. Various microalgal strains such as Chlorella, Dunaliella, Nannochloropsis, Scenedesmus, Neochloris, Nitzschia, Porphyridium, Phaeodactylum, and Isochrysis have been studied for assessing their potential as a biodiesel feedstock (Amaro et al. 2011). Ensuring high lipid accumulation in microalgae is a crucial parameter for making the biodiesel production process economical. Microalgal lipid accumulation can be enhanced by altering the cultivation conditions and nutrients in the media (Singh et al. 2016a). Nitrogen, light, and CO2 stress are widely used strategies for enhancing lipid accumulation in microalgae (Singh et al. 2016b).

Microalgal oils are composed of neutral lipids, polar lipids with some amount of hydrocarbons, sterols, waxes, and pigments (Singh et al. 2014). The neutral lipids are considered as most suitable for the biodiesel synthesis because of their easy conversion to fatty acid alkyl esters (FAAE). In microalgal cells neutral lipids act as the energy storage components and are mainly composed of triglycerides (TAG) and some amount of free fatty acids (FFA). While the polar lipids serve the structural roles (phospholipids in cell membrane) as well as physiological functions such as cell signaling (sphingolipids) (Sharma et al. 2012). Triglycerides and free fatty acids both can be converted into biodiesel via transesterification and esterification process, respectively. However, free fatty acid could cause saponification during the reaction if alkali catalysts are used. Microalgal lipids are known to contain high free fatty acid content.

Biodiesel fuel properties are influenced by the number of carbon atoms in the chain, degree of unsaturation, percentage composition of saturated and unsaturated fatty acid in microalgal lipids. Thus it is important to choose microalgal strains with suitable fatty acid composition for biodiesel synthesis which complies with the international standards (Guldhe et al. 2015b; Singh et al. 2014). Microalgal lipids are composed of saturated, monounsaturated, and polyunsaturated fatty acids. Microalgal lipids have shown C14:0, C16:0, C18:1, C18:2, and C18:3 as major contributing fatty acids (Song et al. 2013). These fatty acids are considered as most suitable for quality biodiesel production. The cultivation conditions and nutrients supplied also have an influence on the fatty acid composition of the microalgal lipids. Table 1 depicts the fatty acid compositions and lipid content of different microalgae used for biodiesel production.

Table 1 Fatty acid profile and lipid content of different microalgae
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3 Transesterification Process

In the process of biodiesel synthesis, triglycerides and methanol are interacted in a reaction called as transesterification or alcoholysis. Methyl esters of fatty acids, i.e., biodiesel and glycerol are the products formed in the transesterification reaction (Cook and Beyea 2000). A catalyst is used to facilitate this reaction via improved rate of reaction and high product yield. Excess alcohol is added to shift the equilibrium of this reversible reaction towards the products side. Alcohols such as methanol, ethanol, propanol, butanol, and pentanol can be used in the transesterification process. Methanol is used more commonly because of its low cost and its physical and chemical advantages (Helwani et al. 2009). NaOH dissolves easily in alcohols and reacts with triglycerides. In a transesterification reaction stoichiometrically, a 3:1 molar ratio of alcohol to triglycerides is needed. Generally the ratio needs to be higher to drive the reaction equilibrium towards maximum ester yield. Biomass-derived fuels share many of the same characteristics as their fossil fuel counterparts. Once formed, they can be substituted in whole or in part for petroleum-derived products. The general reaction mechanism is shown in Fig. 1.

Fig. 1
figure 1

Transesterification of oils to fatty acid alkyl esters

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4 Catalysts

The transesterification reaction can be catalyzed by chemical (alkali and acid) or enzyme catalysts. The chemical alkali catalysts include NaOH, KOH, carbonates, and corresponding sodium and potassium alkoxides such as sodium methoxide, sodium ethoxide, sodium propoxide, and sodium butoxide. Sulfuric acid, sulfonic acids, and hydrochloric acid are commonly used as chemical acid catalysts. Enzyme lipases from various sources are used as biocatalyst for biodiesel synthesis. Alkali-catalyzed transesterification is faster than acid-catalyzed transesterification (Benemann 1997). The catalytic conversion has not been meticulously studied for microalgal biodiesel production. Table 2 shows the various catalysts used for conversion of microalgal lipids to biodiesel.

Table 2 Conversion of microalgal lipids to biodiesel by using various types of catalysts
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4.1 Homogeneous Chemical Catalyst

4.1.1 Homogeneous Acid Catalysts

Homogeneous acid catalysts can produce biodiesel from low cost lipid feedstocks, generally associated with high FFA concentrations (cooking oil and greases, FFA>6%). Microalgal lipids are also known to have high FFA concentrations in the lipids. For acid-catalyzed systems, sulfuric acid, HCl, BF3, H3PO4, and organic sulfonic acids have been used by various researchers (Vyas et al. 2010). Mathimani et al. (2015) comparatively studied the transesterification of Chlorella sp. Lipids using various types of catalyst. In their study they used homogeneous acid (H2SO4), homogeneous alkali (NaOH), heterogeneous acid (Fe2(SO4)3), and heterogeneous alkali (CaO) catalysts. The study showed that the maximum biodiesel yield was detected with the transesterification process catalyzed by homogeneous acid catalyst.

The mechanism of the acid-catalyzed transesterification of oils is depicted in Fig. 2. Mechanism represented here is for monoglyceride, it can be applied to di- and triglycerides. The protonation of the carbonyl group of the ester forms the carbocation (II). The carbocation undergoes a nucleophilic attack of the alcohol, which leads to the tetrahedral intermediate (III), followed by elimination of glycerol to form the new ester (IV), and to redevelop the catalyst H+. According to this mechanism, if water is present in the reaction mixture carboxylic acids can be formed by reacting with carbocation. Thus to avoid the loss of product acid-catalyzed reaction needs to be performed in the absence of water (Schuchardta et al. 1998).

Fig. 2
figure 2

Mechanism of the acid-catalyzed transesterification of oils

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4.1.2 Homogeneous Alkali Catalyst

The most commonly used alkali catalysts for biodiesel synthesis are KOH, NaOH, and CH3ONa (Gemma et al. 2004). The reaction mechanism for alkali-catalyzed transesterification was determined as a three step process. The alkali-catalyzed transesterification is faster than the acid-catalyzed reaction. The mechanism of the base-catalyzed transesterification of oils is shown in Fig. 3. In the first step the base catalyst reacts with the alcohol to produce an alkoxide and protonated catalyst. In the second step nucleophilic attack of the alkoxide at the carbonyl group of the triglyceride leads to a tetrahedral intermediate from which the alkyl ester and the corresponding anion of the diglyceride are generated. The anion of diglyceride deprotonates the catalyst, thus regenerating the active species, which is now available to react with a second molecule of the alcohol, for another catalytic cycle. Diglycerides and monoglycerides follow the same mechanism to form a mixture of alkyl esters and glycerol.

Fig. 3
figure 3

Mechanism of the base-catalyzed transesterification of vegetable oils

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In the alkali-catalyzed transesterification reaction, catalyst is dissolved in methanol by stirring. The mixture of catalyst and alcohol mixture is added to the oil. The reaction mixture is stirred continuously at ambient pressure. After completion of the reaction two liquid phases are produced: ester and crude glycerol. Crude glycerol settles down at the bottom after several hours of settling. After settling is complete, water is added to the reaction mixture followed by stirring, and the glycerine is allowed to settle again. A two step process washing is performed for ester recovery, which needs to be performed with extreme care. First step is water wash and then the acid treatment with stirring. Air is carefully passed through the aqueous layer while gently stirring. This process is continued until the clear ester layer is formed. After settling, the aqueous solution is drained, and water is added again for the final washing (Demirbas 2005; Demirbas 2008). Since base-catalyzed transesterification proceeds faster than the acid-catalyzed reaction and their less corrosive nature than the acidic compounds, industrial processes usually apply base catalysts.

If feedstock oil contains high amount of free fatty acids the alkali catalyst leads to the soap formation which affects the biodiesel yield and also causes problem in the washing steps (Singh et al. 2014). Even when water-free alcohol or oil mixture is used, some water is generated in the reaction mixture by the interaction of the hydroxide with the alcohol. The presence of water leads to the hydrolysis of some amount of ester, with resultant soap formation (Fig. 4). The undesirable saponification reaction hampers the ester yields and makes it difficult to recover the glycerol due to the formation of emulsions (Freedman et al. 1984).

Fig. 4
figure 4

Saponification reaction of the fatty acid alkyl esters produced

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4.2 Heterogeneous Chemical Catalyst

There are numerous heterogeneous catalysts that have been studied for biodiesel synthesis. The heterogeneous catalysts utilized in synthesis of biodiesel are grouped as solid acid and solid base catalyst. Solid acid catalyst includes a wide range of chemicals viz. resins, tungstated and sulfated zirconia, polyaniline sulfate, heteropolyacid, metal complexes, sulfated tin oxide, zeolite, acidic ionic liquids, and others have been used by researchers. Solid base catalysts also include a wide range of catalysts viz. calcium oxide, hydrotalcite (also called layered double hydroxide), alumina, and zeolites (Lam et al. 2010; Sharma et al. 2011). Homogeneous acid and base catalysts used for biodiesel synthesis have several disadvantages, e.g., corrosion of the reactors, metal pipes, storage tanks, and engines. Tedious washing process for their removal is energy intensive and generates wastewater. The heterogeneous catalysts give the advantage of easy separation and thus reuse (Chouhan and Sarma 2011). Reuse of heterogeneous catalyst could improve the economics of the conversion process. Zhang et al. (2012) used KOH/La-Ba-Al2O3 as a heterogeneous catalyst for conversion of microalgal lipids. The reaction was carried out at 60 °C for 3 h and they observed the highest conversion of 97.7% in their study with 25% loading of KOH on modified alumina. To reduce the cost of catalyst several low cost heterogeneous catalysts derived from the waste materials, eggshell, bones, mollusks fish waste, etc., are applied for biodiesel production (Singh et al. 2014). Nur Syazwani et al. (2015) synthesized the CaO catalyst from Angel Wing shell and applied for transesterification of Nannochloropsis oculata lipids. They observed the 84.11% biodiesel yield with 9% catalysts concentration and 1:150 oil to methanol molar ratio in 1 h reaction time. Leaching of the heterogeneous catalysts into the final product, i.e., biodiesel is the major concern. Leached catalyst into the product could hamper the fuel quality as well as its performance in the engines (Singh et al. 2014). A large number of heterogeneous catalysts have been studied for the synthesis of biodiesel from the edible and non-edible plant based oils; however, application of these catalysts on the microalgal based oil is yet to be thoroughly studied.

4.3 Biocatalysts

Enzyme lipase is employed as the biocatalyst for conversion of oils to biodiesel. Lipase is capable of catalyzing both esterification and transesterification process. Thus lipases can be effectively used for conversion of microalgal lipids with high free fatty acid concentration to biodiesel (Guldhe et al. 2015a). Enzyme catalyst provides several advantages over the chemical catalysts. Enzymes are known to possess high selectivity and specificity which lead to high purity product formation. Thus enzymatic transesterification gives high purity biodiesel and by-product glycerol. Less purification steps and minimal use of chemical minimize the wastewater generation unlike in chemical catalyzed biodiesel synthesis. Enzyme catalyst does not require high temperature conditions and can function effectively at atmospheric pressure. Thus the energy input for the enzyme catalyzed reactions is lower than the chemical catalysts (Robles-Medina et al. 2009). Major constraint while using lipases as a catalyst is its high price. Lipase if immobilized can be separated from the reaction mixture and reused for several batches of the conversion process. Novel immobilization techniques such as cross-linked protein coated microcrystals, magnetic support particle, and nanofiber are used for lipases to improve their catalytic activity, reuse potential, and stability in solvents (Guldhe et al. 2015a). The reuse potential of biocatalyst can reduce the cost of catalytic conversion. The activity of lipases can be inhibited by the presence of excessive alcohol (<3 moles). Thus several researchers have suggested the stepwise addition of methanol in the reaction mixture (Fukuda et al. 2001; Guldhe et al. 2015b). Stoichiometrically 3 moles of methanol is needed in the transesterification reaction for 1 mole of triglyceride. In lipase catalyzed transesterification 1 mole equivalent of methanol with respect to oil is added thrice after periodic interval during the reaction. Enzyme catalysis can be carried out in two ways, viz., using immobilized extracellular lipases and immobilized whole cells (intracellular) producing lipase. Xiong et al. (2008) used the Candida sp. 99–125 sp. lipase for the transesterification of Chlorella protothecoides lipids and observed 98.15% biodiesel conversion. The application of biocatalyst for conversion of microalgal lipids to biodiesel can make the process greener.

5 Acyl Acceptors Used in Transesterification of Microalgal Lipids

Transesterification is the common used process to convert triglycerides into biodiesel. This consists of the reaction of triglyceride and an acyl acceptor. Carboxylic acids, alcohols, and other ester can be used as an acyl acceptor. Glycerol is produced in transesterification when alcohol is used as an acyl acceptor while triacylglycerol is produced when ester is used as an acyl acceptor. Several acyl acceptors have been studied by the researchers for transesterification process. Methanol is the most widely used acyl acceptor in transesterification process (Helwani et al. 2009). Methanol is short chain alcohol which leads to faster reaction rate and also its inexpensive nature makes it favorable acyl acceptor at an industrial scale biodiesel production process. The major drawback of methanol is its toxic nature which raises the concerns regarding environmental and accidental risks. Methanol as an acyl acceptor is more toxic for lipase activity compared to ethanol. Use of ethanol as an acyl acceptor can improve the catalytic performance of lipase and also its reuse potential (Raita et al. 2010). In addition, ethanol can be produced from renewable sources via fermentation, which makes the process of biodiesel production greener. Only few studies report use of ethanol as acyl acceptor. The literature unanimously recommends a stepwise addition of alcohols to reduce their toxic effect towards enzyme catalyst (Guldhe et al. 2015a). Further research is needed for exploring the potential of other compounds as acyl acceptor for transesterification reaction.

6 Solvents Used in Transesterification of Microalgal Lipids

The feedstock oil and alcohol are immiscible and thus slow down the reaction rate. To overcome this problem researchers have suggested the use of solvent in the transesterification reaction. Solvents increase the mass transfer rate which eventually results in better reactant interactions and fast reaction rate (Abbaszaadeh et al. 2012). Various solvents have been applied in the transesterification. Hexane, tetrahydrofuran, and diethyl ether are most popular solvents for transesterification reaction in biodiesel synthesis. Lam and lee (2013) investigated the effect of various solvents (hexane, ethanol, tetrahydrofuran, toluene, methyl acetate, ethyl acetate, and chloroform) on the transesterification of C. vulgaris oil using sulfuric acid as a catalyst. Among the various solvents studied by them tetrahydrofuran significantly improves the reaction rate and also reduces the methanol and catalyst amounts needed for reaction.

The solvents need to be removed from the biodiesel as well as glycerol after the completion of the reaction. Solvents are readily available at low cost, thus its use does not drastically affect the economics of the production process. However, high volatility, toxicity, potential hazards, and environmental risk raise concerns regarding their usage. To overcome these concerns recently ionic liquids have been employed as the solvent in transesterification reaction. Ionic liquids are termed as green solvents because of their properties such as negligible vapor pressure, high solubility, and tunable as per reaction requirement (Mohammad Fauzi and Amin 2012). Much more attention needs to be given on the novel solvents such as ionic liquids for their application in microalgal biodiesel production process.

7 In-Situ Transesterification

In-situ transesterification couples the extraction of lipids and transesterification via catalysis together. In-situ transesterification is alternative to the conventional process, which has the potential of reducing the processing steps and the overall conversion cost. The in-situ process aids the conversion of the oil to biodiesel directly from the oil bearing biomass. In-situ transesterification process eliminates the solvent extraction step. This technique thus reduces the requirement of solvents used for extraction step (Ehimen et al. 2010). This process is also gaining recognition as a lipid measurement procedure for algae (Laurens et al. 2012). In-situ transesterification can be done by using dry or wet microalgal biomass. Recently microwave and sonication assisted in-situ transesterification are studied for increasing the biodiesel yield. Tran et al. (2012) studied the in-situ transesterification of wet C. vulgaris biomass catalyzed by Burkholderia lipase. They observed 97.3% biodiesel yield when sonication pretreated biomass was subjected to in-situ transesterification in hexane as a co-solvent. This technique has shown potential to reduce the number of processing steps, amount of organic solvent used, and thus the overall production cost of biodiesel synthesis.

8 Process Intensification by Microwave and Ultrasound

Microalgal biodiesel is still far from commercial realization because of the high cost of production. Process intensification to reduce the reaction time and increase the yields could aid in reducing the biodiesel production cost. Catalytic conversion of oils to biodiesel can be intensified by using microwave or ultrasound techniques. The microwave or ultrasound assisted lipid conversion processes offer the advantages of shorter reaction time and higher yields over the conventional heating processes. The transesterification reaction assisted by microwave and sonication has been extensively investigated with vegetable oils in recent years to enhance the biodiesel yield. Microwave irradiation directly delivers energy to the reactants in the transesterification reaction. Thus the microwave assisted reaction completes in shorter time because of the effective heat transfer. The mass transfer of reactants in a sonicator is about 10 times faster than the conventional mode of stirring (Gole and Gogate 2012). Microwave and sonication techniques can be effectively coupled with in-situ transesterification of microalgal biomass (Guldhe et al. 2014). In in-situ transesterification process these intensification techniques serve dual purpose of breaking the cell wall as well as effective mass transfer. These process intensification techniques are, however, associated with high energy consumption. Further investigation is needed to improve the efficiency of using microwave or sonication for transesterification of microalgal lipids.

9 Challenges and Future Prospective of Catalytic Conversion of Microalgal Lipids

Biodiesel from microalgae is identified as promising future transport fuel. However, at present the high production cost is the major bottleneck in its commercial scale production. Conversion of microalgal lipids to biodiesel is a key step in production process. Lot of attention has been provided on the up-stream steps such as strain selection, cultivation, and lipid enhancement in microalgal biodiesel production process. There is need for thorough research on the microalgal lipids conversion process using various catalysts. Varying lipid quality, low yields, long reaction time, scaling-up, and achieving desired quality product are some of the major challenges in catalytic conversion process of microalgal lipids. High free fatty acid content in microalgal lipids advocates the use of acid or enzyme catalyst for conversion reaction. Numerous heterogeneous catalysts have been investigated for conversion of vegetable and other feedstock oils. This promising group of catalysts needs to be thoroughly studied for its application in microalgal biodiesel production. In-situ transesterification has shown potential to reduce the production cost by avoiding the extraction step. Low product yields in in-situ transesterification process need attention from the researchers to make this technology efficient. Process intensification by microwave and ultrasound techniques has shown potential to improve the yields in conventional as well as in-situ transesterification processes. Scaling-up of the efficient conversion process is also a challenging task. Comprehensive techno-economic and environmental risk assessment studies need to be conducted for competent conversion technologies for microalgal biodiesel production. Efficient and economically viable conversion technologies will lead the microalgal biodiesel production towards sustainability and greener future.