Enzymatic Biodiesel Production: Challenges and Future Perspectives

Erika C. G. Aguieiras Eliane P. Cipolatti Martina C. C. Pinto Jaqueline G. Duarte Evelin Andrade Manoel Denise M. G. Freire


The development of more sustainable and environment-friendly processes, based on principles of a circular economy, is a current need for the most different productive sectors, including the biofuels sector. Over recent years, the production of biodiesel through biotechnological routes has aroused enormous interest from academia and industry (Figure 13.1). Based on the available data, it has been observed that work regarding biodiesel and enzymes has exhibited a similar rising tendency. In this scenario, the use of lipases as biocatalysts to carry out such biotransformation is an environmentally attractive and, often, economically viable alternative.

  • — web of science
  • (keywords: biodiesel and enzyme)
  • — — web of science
  • (keywords: biodiesel)

patent inspiration

  • (keywords: biodiesel and enzyme) patent inspiration
  • (keywords: biodiesel)
Evolution of biodiesel studies over recent years, based on two different databases

FIGURE 13.1 Evolution of biodiesel studies over recent years, based on two different databases: the Web of Science and patent inspiration.

Lipases (‘glycerol ester hydrolases’, E.C. are hydrolytic enzymes that catalyze the hydrolysis reactions of ‘triglycerides' (TAG), producing ‘diglycerides’ (DAG), ‘monoglycerides’(MAG), ‘free fatty acids'(FFA), and‘glycerol’. In aquo-restricted environments, these enzymes are able to catalyze synthesis reactions, including esterification and transesterification (Koskinen and Klibanov, 1996). Because of the high catalytic performance of these proteins, lipases are the third most commercialized biocatalysts, being largely used in detergent, food, paper and cellulose, animal feed, and the pharmaceutical industries. The market for microbial lipases was valued at USD425 million in 2018 and is projected to grow at 6.8% per year, reaching USD590 million by 2023 (Almeida et al., 2020).

The versatility of such enzymes, as well as the catalytic reaction under mild process conditions, the possibility of their reuse, the facility of separation steps, and the environmentally friendly process of using enzymes, has aroused the interest of researchers regarding its application in biodiesel production (Hama et al., 2018). Moreover, the robustness of lipases in the presence of FFA in feedstocks, since such enzymes catalyze both esterification and transesterification reactions, makes these proteins ideal to produce esters from partially hydrolyzed raw materials (acidity > 0.5%).

The literature reports the use of lipases from different sources including animals, vegetables, and microbial-based sources for biodiesel synthesis (Aguieiras et al., 2014; Rial et al., 2020; Sharma et al., 2001). However, microbial lipases are the most used due to their easy large-scale production, high versatility regarding distinct selectivity, and the possibility of genetic manipulation which aims at their production by heterologous expression with high productivity.

In this context, through ‘protein engineering’ tools, including directed evolution and rational design, it is possible to acquire lipase variants that are highly tolerant under industrially severe conditions, such as the presence of solvents, and high

267 temperature and pressure, making it possible to design an ideal biocatalyst for a given process (Dior et al., 2015; Hama et al., 2018).

Metagenomics is another tool that enables the extraction of all microbial genomic DNA from certain environmental habitats, the construction of a metagenomic library, and the screening of novel thermo and solvent-tolerant lipase from the uncultivable components of microbial environments (Almeida et al., 2020; Hama et al., 2018; Sahoo et al., 2017).

The first paper reporting the use of lipase for biodiesel synthesis was described in 1990 by Mittelbach (1990). Since then, the number of publications and patents involving the application of lipases in biodiesel synthesis has grown. Most work has focused on some topics that may lead to high conversions to oil in ‘fatty acid methyl esters’ (FAME) and ‘fatty acid ethyl esters’ (FAEE). such as: the development of new biocatalysts aiming to diminish their cost and increase their activity and stability; the search for low-cost raw materials; the study of different reactor configurations; and the evaluation of the reaction parameters of temperature, enzyme concentration, the molar ratio of reagents, and the presence of a solvent. However, not only the scale-up of the biodiesel process but also work that focuses on eco-economic analyses related to enzymatic production of biodiesel is also scarce.

This chapter reviews the main aspects and challenges related to enzymatic biodiesel production and points out some future perspectives towards turning this process into an industrial reality. The development of new biocatalysts and research involving alternative feedstocks are also illustrated. A brief discussion regarding possible routes for obtaining enzymatic biodiesel, dependent on the use of raw materials, is presented. Attempts to scale up the enzymatic process, as well as work on economic analysis, are also presented and discussed.

Technological Challenges for Enzymatic Biodiesel Production

Commercial lipases used in biodiesel production are mainly produced by heterologous expression and commercialized by companies such as Novozymes, Amano, Gist Brocades, etc. (Sharma et al., 2001; Yan et al., 2014a). Lipase B from the yeast ‘Candida antarctica’ (CALB) is the most widely used lipase (Aarthy et al., 2014). Lipases from filamentous fungi, such as ‘Rhizomucor miehei lipase’ (RML) (Aguieiras et al., 2017b; Rodrigues and Fernandez-Lafuente, 2010), Rhiz.opus oryzae lipase (ROL) (Canet et al., 2014), and ‘Thermomyces lanuginosus lipase’ (TLL) (Fernandez-Lafuente, 2010) have also been largely used. The most used bacterial lipase is ‘Burkholderia cepacia lipase’, formerly known as Pseudomonas cepacia (Amano Lipase PS) (Kumar et al., 2020; Nelson et al., 1996). Many of these enzymes are also commercially available in their immobilized form by Novozymes as Novozym 435 (CALB immobilized on a macroporous acrylic resin), ‘Lipozyme RM IM' (RML immobilized on an anionic resin), and ‘Lipozyme TL IM' (TLL immobilized on a gel of granulated silica) (Nielsen et al., 2008; Robles-Medina et al„ 2009).

Although enzymatic biodiesel production is widely reported in the literature, some drawbacks, including the high cost of the biocatalysts, enzyme deactivation, and lower reaction rates, when compared to conventional processes, still limit the application of lipases to produce commodity products, such as biodiesel

(Freire et al., 2011). Studies to find solutions for such problems are focused on three ways: (1) biocatalysts, (2) reaction conditions, and (3) reactor configurations. Some of these topics are discussed more deeply in the next sections.


The academic and industrial sectors have made an effort to develop higher active and stable biocatalysts to be subjected to different reaction conditions, whether application is at the small or large scale (Hartmann and Jung, 2010). Although the search for an ideal biocatalyst is one of the principal challenges related to the enzymatic production of biodiesel, the number of industrial uses of enzymes has significantly increased in recent years, mainly due to advances in protein engineering technology and environmental and economic demands.

The use of biocatalysts for biodiesel production can be divided into four main categories: free enzymes, ‘immobilized lipases’, ‘whole-cells’ and ‘dry fermented solids’ (DFS) obtained by ‘solid-state fermentation’ (SSF), as illustrated in Figure 13.2. Soluble enzymes have the advantages of a lower purchase cost, compared to immobilized enzymes, and practically no diffusional limitations, which improves the mass transfer of substrates and products (Andrade et al., 2019). However, free lipases, in soluble or powder formulations (obtained by freeze-drying), have the disadvantage of poor operational stability and easy deactivation. In addition, the formation of enzyme aggregates in organic media and the safety concerns regarding the use of enzyme powders, mainly on large-scale processes, are other issues related to the use of free lipases (Freire et al., 2011).

Distinct biocatalysts used in biodiesel production

FIGURE 13.2 Distinct biocatalysts used in biodiesel production: (a) free lipase; (b) immobilized lipase: (c) whole-cell biocatalyst; (d) a dry fermented solid (here presented as the DFS obtained through growth of Rhizomucor iniehei by SSF in cotton seed cake).

In this way, many technologies, comprising the production of more robust and low-cost biocatalysts, emerged which aimed at the development of more sustainable and economically viable processes.

Recent Advances in Lipase Immobilization

Enzyme immobilization technology is used as an important tool to improve enzyme properties (activity; stability, considering both operational and storage stabilities; specificity; and selectivity) and to permit the reuse of the biocatalysts in distinct reaction cycles, since the biocatalyst is insoluble on reaction media (Boudrant et al., 2020; Hartmann and Jung, 2010). Because of the recovery of the enzymes, it is possible to reduce the cost of the process, one of the main issues that limit the industrial production of biodiesel through enzymatic routes (DiCosimo et al., 2013). It is known that enzyme immobilization can occur in several ways, such as adsorption, covalent bonding, and encapsulation, as well as the association of these techniques.

Immobilized lipases have received special attention in the biotechnology field for biodiesel production (Zhong et al., 2020). Such biocatalysts are usually more stable under more severe reaction conditions (conditions in which free enzymes could not be active). Moreover, the use of immobilized biocatalysts reduces the number of reaction steps and avoids the continuous washings needed when biodiesel is synthesized through a chemical route, avoiding the generation of tons of effluent (Zhao et al., 2015).

In this context, the design of new supports and immobilized enzyme preparations is a relevant area of modern sciences and technologies (Cipolatti et al., 2014; Manoel et al., 2016; Pinto et al., 2014; Zaitsev et al., 2019). An emerging area, generically called ‘support engineering’, comprises the development of new polymeric materials in order to interact with specific enzymes, resulting in maximum performance biocatalysts. It is important to highlight that some features are required to use a material as support for enzymatic immobilization, such as insolubility in the substrate and product phases, mechanical stability, porosity, and chemical resistance. The biocatalysts’ performance also depends on numerous factors, including support material, enzyme immobilization method, and the type of enzyme (Cipolatti et al., 2018, 2019; Manoel et al., 2016; Pinto et al., 2014).

Some studies have been conducted using natural polymers as supports for lipase immobilization (such as chitin, chitosan, gelatin, and cellulose). The employment of nanometric materials, including nanofibers, mesoporous nanocarriers, and magnetic nanoparticles, have been explored for the synthesis of distinct ‘nanobiocatalysts’. Another polymerization technique, simultaneous suspension and emulsion polymerization reactions, have also been used for the synthesis of new core-shell supports, once it allows the production of porous micrometric particles and the functionalization of such particles, without the necessity of additional reaction steps (Cipolatti et al. 2018, 2019; Manoel et al., 2016; Pinto et al., 2014). For instance, Cipolatti et al. (2018) produced distinct core-shell supports, exhibiting different compositions, for the immobilization of CAL-B. The biocatalysts were used on esterification reactions aiming at the synthesis of biofuels.

TABLE 13.1

Biodiesel Synthesis Using Immobilized Lipases






Rate (%)



Candida antárctica (fraction B)

Polyporous magnetic cellulose

Yellow horn seed oil


60°C, 2 h. pH 7.5

Zhang et al.


Porcine pancreas

p-nitrobenzyl cellulose xanthate

Soybean oil


2 h. 40°C, pH 7

Rial et al.


Pseudomonas cepacia

Polyvinyl alcohol/ sodium alginate

Castor oil


50°C, 24 h

Kumar et al.


Rhizopus oryzae

Alginatepolyvinyl alcohol beads

Sludge palm oil



Muanruksa and Kaewkannetra (2020)

Rhizopus oryzae

Fe,O4 superpara-magnetic nanoparticles

Oil from Chlorella vulgaris


45°C,24 h

Nematian et al.


Candida antárctica B and Rhizomucor miehei (coimmobilized)


Palm oil


  • 35.6°C,
  • 33.5 h

Shahedi et al.


Obviously, many factors affect the use of lipases for biodiesel production. Table

13.1 summarizes some important factors related to the most recent studies in the area of immobilized lipases. It can be seen that the conversion rate depends on the substrate, the reaction conditions, the type of enzyme, and the support composition. It is important to highlight that the factors that directly interfere with biodiesel synthesis are not restricted to the ones exhibited in Table 13.1.

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