**1. Introduction**

The transport sector is currently facing an unprecedented challenge. On the one hand, fossil fuels represent more than 95% of the energy employed in this sector, increasing day by day. However, as fossil fuels have a finite nature, they cannot cope with this demand indefinitely. On the other hand, the control of greenhouse gas emissions, including CO2, is mandatory for environmental purposes. Thus, an smooth transition from the current scenario, in which diesel engines work mainly with fossil fuels, to another in which these engines will work mainly with renewable biofuels would allow to respond the increasing demand on fuels, as well as to partially solve the environmental issues [1,2].

In fact, biofuels have been postulated as the best option to replace fossil fuels, since to date, electric motors or vehicles capable of using fuel cells cannot compete ye<sup>t</sup> with explosion or combustion engines, especially in the field of heavy trucks [3], aviation [4,5], or the shipping sector [6]. Therefore, research on renewable fuels capable of replacing fossil fuels and allowing current engines to operate without any modification constitutes a first order priority [7,8]. Among biofuels, biodiesel is considered the best option to replace fossil fuels in compression ignition diesel engines, since no modifications have

to be performed [8–11]. Industrial production of biodiesel is currently carried out by homogeneous alkali-catalyzed transesterification of vegetable oils with methanol [12]. After the reaction, biodiesel is repeatedly washed with water to remove glycerol and soaps [13]. In this process, the generation of glycerol as byproduct is a major issue for both, the high amount of glycerol generated (10% by weight of the total biodiesel produced) and the high amount of water employed for removing it [14]. Thus, to solve this drawback, several alternative methods are being investigated.

One option is the production of glycerol derivatives, with rheological properties like methyl esters of fatty acids, during the same transesterification process. This process allows its dissolution in the biofuel and/or in the fossil diesel, increasing the yield of the process and also avoiding the separation of glycerol [15,16]. To do that, methanol is replaced by other donor agents, such as ethyl (or methyl) acetate or dimethyl carbonate. Different catalysts have been investigated, including several lipases [17,18]. As a result of these transesterification reactions, a mixture of three molecules of FAME or FAEE (fatty acid ethyl ester) and one of glycerol carbonate or glycerol triacetate (triacetin) are obtained [19,20].

Other valuable option is the production of a biofuel, with similar physicochemical properties as biodiesel, which integrates glycerol in the form of monoglyceride. In this field, our Research Group has an extensive background, specifically in the production of Ecodiesel, a biofuel obtained by a selective 1,3-regiospecific enzymatic transesterification of the triglycerides (Scheme 1), so that a mixture of two parts of FAEE and one part of MG is obtained [21]. The experimental conditions of the enzymatic process to produce Ecodiesel are much milder than those required for a conventional homogenous process. In addition, the atomic efficiency of the process is very high, since the yield of the process is practically 100%, avoiding, then, any process to eliminate impurities from the biofuel obtained. Last but not least, as a reagent, ethanol is used, a cheaper compound than dimethyl carbonate or ethyl acetate, both described as alternatives for obtaining biofuels that integrate glycerin as soluble compounds in the biofuel.

**Scheme 1.** Ecodiesel synthesis by 1,3-selective ethanolysis of triglycerides, which integrates the glycerol as monoglyceride.

The Ecodiesel synthesis was patented using a pig pancreatic lipase PPL [22], although remarkable results have been also obtained over different microbial lipases [23,24]. To optimize the different reaction conditions, such as the effect of temperature, the pH, the oil/alcohol molar ratio, etc. statistical analysis of variance (ANOVA) and response surface methodology (RSM) have been previously employed in Ecodiesel production [25].

Anyhow, despite lipases can be employed as catalyst to perform the Ecodiesel synthesis, their high production cost is still a huge limitation for their use at industrial scale. To overcome this, lipases must be able to be reused in subsequent reactions, being a viable option its immobilization on a support. In this sense, we have recently reported the immobilization of Lipozyme RM IM, a *Rhizomucor miehei* lipase, on a macroporous anion exchange resin and its use as heterogeneous biocatalyst in the selective transesterification of sunflower oil with ethanol [26]. Despite the good results obtained, the low density of the polymer resin lipase makes its recovery very difficult, needing a centrifugation operation at 3500 rpm for 5 minutes. Furthermore, a maximum of six reuses can be attained before the loss of the

activity. From an economical point of view, six reuses are not enough if we take under consideration the high price of the lipase.

Hence, in this research, two different aspects have been addressed. On one hand, to avoid the centrifugation process for recovering the biocatalyst, several immobilization methods on inorganic supports have been evaluated. For that purpose, two different cheap inorganic materials, sepiolite and silica, have been evaluated as supports using covalent immobilization and/or physical adsorption.

On the other hand, several reaction parameters have been studied by ANOVA and RSM, to have better insights into the production of Ecodiesel over the *Rhizomucor miehei* lipase. We consider this statistical method very helpful because, as we previously observed employing other enzymes, during the production of Ecodiesel, the properties of the enzymes change depending on these reaction parameters, influencing the measured reaction rates [21,25,27]. In this study, we have evaluated the influence of the amount of lipase, the amount of 10 N NaOH and the oil/ethanol molar ratio in the production of Ecodiesel.

## **2. Materials and Methods**
