**3. Etherification of glycerol from biodiesel production with company´s own residues**

The need for renewable energies in general and biodiesel in particular, indicates that optimis‐ ing the production process is of vital importance. Biodiesel production generates *ca*. 10% of glycerol as a subproduct which has led to a fall in the glycerol prices, making the search for other industrial applications a neccesity. Amongst all possible processes to increase the value of glycerol, etherification is one of the most promising, since glycerol ethers can be used as such or with slight modification as fuel additives [20]. Other important uses are found in cosmetics, food additives, monomers for polimerisation processes *etc*. [21].

This research was undertaken with the aim to use Acesur-Tarancon´s own subproducts to prepare catalysts to transform the company´s glycerol (from their biodiesel plant) to diglicerol ethers. The catalysts prepared in this way are in fact Ecomaterials and their origin makes them competitive with commercial ones. Production of ethers with more than three glycerol molecules competes with diethers and thus control of the selectitivy is important, especially keeping in mind that the diglycerol ethers (Figure 6) are produced at short reaction times, which gives the process an added value [20, 22].

**Figure 6.** Glycerol pathway to di- and triglycerols in reference 23.

These results indicated that the carbon prepared from beer residues had better adsorption capacities for cleaning the DMG wastewater than the commercial one, especially with regards to the high molecular weight substances. This can be related to the different textures, since the commercial carbon has smaller pores that can not easily accommodate the high molecular weight substances. The chemical oxygen demand (COD) of the original and treated wastewa‐

( 1 2 )

**COD % Reduction**

**4**

0

where C: concentration of Fe(II) sulphate and ammoniumin mol/L, V0: volume in mL before dilution, V1: volume in mL of Fe(II) sulphate and ammonium solution for blank analysis, V2: is the volume in mL of Fe(II) sulphate and ammonium solution for assay, 8000 is molar mass in mg/L of ½ O2. The COD results are in mg O2/L. The value of the method has been checked with a 0.425 g of potassium hydrogenphtalate (KC8H5O4), dried at 105°C, diluted in 1000 mL distilled water with a COD standard value of 500 mg O2/L (+/- 20). Variabilities in COD analyses were less than 2 % [18]. The results obtained for the COD reduction of the wastewater, with the different materials are included in Table 3 (Percent reduction of COD after room temper‐ ature wastewater treatment with adsorbents until constant COD (usually *ca*. 60 minutes).

*CVV V* ´´ -

ters was studied according to the Spanish UNE 77004, equivalent to ISO 6060:1989:

800

DQO :

**Comercial (Fluesorb B)**

**Table 3.** Percent COD reduction of DMG wastewater with adsorbents.

developed [19].

192 Agroecology

BBM2 5 BBM4 10 BBM6 18 RH2 17 RH4 67 RH6 73

The high effectiveness of the residue derived materials compared with the commercial carbon should be noted. The material with greater cleaning ability was RH6 (73% reduction), which allowed a water with COD of 960 mg O2 /L. Comparing the textural data with COD determi‐ nations, it can be said that there was a direct correlation between the pore diameter of the solids and their COD reduction capacity. Thus, wastewater treatment with residue derived materials has been shown to be an economical and environmentally sound process that should be further

Etherification of glycerol with acid catalysts was found to be difficult to control. However, the catalytic transformation of glycerol into ethers, carried out with basic catalysts allows more controllable results. Furthermore, the use of heterogeneous catalysts *i.e.* alkaline and alkaline earth oxides, present in the residue derived materials form Acesur, compared to homogeneous bases is gaining interest since they are easily separated from the reactants and products for reuse with the corresponding economic benefits [24]. A bibliographic search showed Barrault ´s work describing caesium oxide catalysts that achieved medium conversions with selectivi‐ ties to di- and triglycerols, depending mainly on the reaction time [25]. Also Ruppert describes a reaction carried out on alkaline-earth oxides at 220 °C for 20 h giving rise to higher glycerol conversions on the more basic catalysts: 5 % (MgO), 58 % (CaO), 80 % (SrO) and 80 % (BaO) [26].

The oligoglycerols are gaining more and more interest as products used in cosmetics, foodadditives or lubricants [27]. Short overviews about the synthesis of glycerol oligomers from di- to pentaglycerol have been published by Rollin *et al* [28]*.* Generally oligoglycerols are produced using basic homogeneous catalysis, but lately increased attention has been paid towards the heterogeneously catalysed processes. Despite a lower activity heterogeneous catalysts reveal many advantages: firstly, the separation of the catalyst and secondly, by carrying out the reaction in the absence of solvent, in this work only filtering the catalyst was needed, with evident economic and environmental advantages.

The conditions used for glycerol etherification were chosen with basic catalysis, since the sunflower oil production agriresidue derived materials (RP45), given their composition of alkaline (26 % potassium) and alkaline-earth cations (5 % magnesium and 7 % calcium) are basic in nature. TGMS of adsorbed acetic acid indicated that RP45 contained basic centres of low (100-200 °C), medium (200-500 °C) and high basicity (550-650 °C) (Figure 7) (see procedure for basicity measurement in reference 14) and can catalyse Knoevenagel condensation reactions [29].

**Figure 7.** Analysis of basicity of RP45 by TG-MS of acetic acid decomposition.

The reactions were carried out in abscense of solvent, under inert atmosphere to limit over‐ oxidation, controlling temperature and time of reaction to optimise the economics [30, 31]. The work described here was carried out to produce diglycerol ethers in the absence of solvent, under inert atmosphere to avoid overoxidation, using catalysts derived from sunflower oil production residues, which have medium and high basic strengths. The analysis of reaction products was carried out by GC-MS (conditions as in reference 14).

Optimisation of the reaction conditions (Figure 8) showed that with 240°C, under inert atmosphere (nitrogen flow) and a ratio catalyst/glycerol = 1/50, after 4 h the conversions of glycerol and selectivities to diglycerols were optimum. At lower temperatures the conversions were low and with higher temperatures the selectivity to diglycerols decreased due mainly to uncontrolled formation of polyglycerols and oxidised compounds (mainly glycolic and glyceric acids).

**Figure 8.** Conversions and selectivities of glycerol to diglicerol at different reaction temperatures.

Main compounds found in the reaction carried out in this work are summarised in Figure 9.

**Figure 9.** Main diglycerol ethers found in the present work**.**

di- to pentaglycerol have been published by Rollin *et al* [28]*.* Generally oligoglycerols are produced using basic homogeneous catalysis, but lately increased attention has been paid towards the heterogeneously catalysed processes. Despite a lower activity heterogeneous catalysts reveal many advantages: firstly, the separation of the catalyst and secondly, by carrying out the reaction in the absence of solvent, in this work only filtering the catalyst was

The conditions used for glycerol etherification were chosen with basic catalysis, since the sunflower oil production agriresidue derived materials (RP45), given their composition of alkaline (26 % potassium) and alkaline-earth cations (5 % magnesium and 7 % calcium) are basic in nature. TGMS of adsorbed acetic acid indicated that RP45 contained basic centres of low (100-200 °C), medium (200-500 °C) and high basicity (550-650 °C) (Figure 7) (see procedure for basicity measurement in reference 14) and can catalyse Knoevenagel condensation

The reactions were carried out in abscense of solvent, under inert atmosphere to limit over‐ oxidation, controlling temperature and time of reaction to optimise the economics [30, 31]. The work described here was carried out to produce diglycerol ethers in the absence of solvent, under inert atmosphere to avoid overoxidation, using catalysts derived from sunflower oil production residues, which have medium and high basic strengths. The analysis of reaction

Optimisation of the reaction conditions (Figure 8) showed that with 240°C, under inert atmosphere (nitrogen flow) and a ratio catalyst/glycerol = 1/50, after 4 h the conversions of glycerol and selectivities to diglycerols were optimum. At lower temperatures the conversions were low and with higher temperatures the selectivity to diglycerols decreased due mainly to uncontrolled formation of polyglycerols and oxidised compounds (mainly glycolic and

needed, with evident economic and environmental advantages.

**Figure 7.** Analysis of basicity of RP45 by TG-MS of acetic acid decomposition.

products was carried out by GC-MS (conditions as in reference 14).

reactions [29].

194 Agroecology

glyceric acids).

S-4700 type I, Japan). (Figure11).

The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/ Glycerol = 50 [32]. The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 °C), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to over oxidation and triglycerols [33]. The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/Glycerol = 50 [32]. The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 ºC), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to over oxidation and triglycerols [33].

**Figure 10.** a) Homogeneous reaction of glycerol with NaOH (15 min glycerol, 18-20 minutos cyclic diglycerols). b) Activated RP45 (4 h reaction, 5.7 min: glycolic acid, 7.8 min: glyceric acid, 15 min: glycerol, 18-20 min: cyclic diglycerols, 21 min: linear diglycerols, 23 min: triglycerols). **Figure 10.** a) Homogeneous reaction of glycerol with NaOH (15 min glycerol, 18-20 minutos cyclic diglycerols). b) Acti‐ vated RP45 (4 h reaction, 5.7 min: glycolic acid, 7.8 min: glyceric acid, 15 min: glycerol, 18-20 min: cyclic diglycerols, 21 min: linear diglycerols, 23 min: triglycerols).

Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material *in situ* to optimise its activity as a basic catalyst. This was done by heating to 500ºC, reaching conversions close to 30 % after 6 h of reaction with selectivity to linear diglycerols close to 50 %. Also, the use of an inert atmosphere was necessary to avoid over oxidation [34]. The reactions carried out with RP47 and RP48 gave very low conversions as corresponds to their smaller active surfaces, since they were prepared at 700 ºC and 850 ºC respectively (RP47: 8 h, 5 % conversion, 75 % linear diglicerol, 25 % cyclic, RP48: 8 h, 2 % conversion, 50 % cyclic, 50 % linear) [20, 23, 31]. **4. Lipases immobilised on materials prepared with agriresidues derived from rice production.**  Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycerides to diglycerides, monoglycerides, Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material *in situ* to optimise its activity as a basic catalyst. This was done by heating to 500°C, reaching conversions close to 30 % after 6 h of reaction with selectivity to linear diglycerols close to 50 %. Also, the use of an inert atmosphere was necessary to avoid over oxidation [34]. The reactions carried out with RP47 and RP48 gave very low conversions as corresponds to their smaller active surfaces, since they were prepared at 700 °C and 850 °C respectively (RP47: 8 h, 5 % conversion, 75 % linear diglicerol, 25 % cyclic, RP48: 8 h, 2 % conversion, 50 % cyclic, 50 % linear) [20, 23, 31].

#### preparation, hydrolysing the grease in milk or production of pharmacological substances. In the human body these substances are important since they facilitate fats adsorption. Lipase immobilisation is of interest since it allows their reuse and increases their resistance to inactivation. For industrial **4. Lipases immobilised on materials prepared with agriresidues derived from rice production.**

applications, several properties are important *i.e.* mechanical strength, chemical and physical stability, hydrophobic/hydrophilic

fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk

character, amount of immobilised enzyme and cost. The use of agroindustrial residues to prepare supports for immobilisation of enzymes can reduce the cost and therefore extend the use of lipases to an industrial scale, since these materaials are cost effective if the technology to make them competitive with commercial ones is developed. In this work the materials used to support enzymes where derived from rice husk (RH) and sunflower (RP) industrial production [13, 35]. The thermal stabilities of the residues in air were analysed by thermal techniques (TGA–DTA) on a Netzsch 409 EP Simultaneous Thermal Analysis device. Approximately 20–30 mg of powdered samples were heated in an air stream of 75 mLmin<sup>−</sup><sup>1</sup> at a heating rate of 5 °Cmin<sup>−</sup><sup>1</sup> from ambient to *ca.* 1000°C, using α-alumina as a reference. The termal data were used to design the controlled thermal treatments to produce agriresidue derived materials at temperatures between 500-700 ºC, where the organic matter had been decomposed. The microstructure of the agriresidue derived materials was observed by scanning electron microscopy (SEM) (Hitachi Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycer‐ ides to diglycerides, monoglycerides, fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk preparation, hydrolysing the grease in milk or production of pharma‐ cological substances. In the human body these substances are important since they facilitate fats adsorption.

**Figure 11.** SEM images of rice production derived materials. a) RH47 b) RH26

**a) b)** 

a) b)

at a heating

Lipase immobilisation is of interest since it allows their reuse and increases their resistance to inactivation. For industrial applications, several properties are important *i.e.* mechanical strength, chemical and physical stability, hydrophobic/hydrophilic character, amount of immobilised enzyme and cost. The use of agroindustrial residues to prepare supports for immobilisation of enzymes can reduce the cost and therefore extend the use of lipases to an industrial scale, since these materaials are cost effective if the technology to make them competitive with commercial ones is developed. In this work the materials used to support enzymes where derived from rice husk (RH) and sunflower (RP) industrial production [13, 35]. Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycerides to diglycerides, monoglycerides, fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk preparation, hydrolysing the grease in milk or production of pharmacological substances. In the human body these substances are important since they facilitate fats adsorption. Lipase immobilisation is of interest since it allows their reuse and increases their resistance to inactivation. For industrial applications, several properties are important *i.e.* mechanical strength, chemical and physical stability, hydrophobic/hydrophilic

**Figure 10.** a) Homogeneous reaction of glycerol with NaOH (15 min glycerol, 18-20 minutos cyclic diglycerols). b) Activated RP45 (4 h reaction, 5.7 min: glycolic acid, 7.8 min: glyceric acid, 15 min: glycerol, 18-20 min: cyclic diglycerols, 21 min: linear

Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material *in situ* to optimise its activity as a basic catalyst. This was done by heating to 500ºC, reaching conversions close to 30 % after 6 h of reaction with selectivity to linear diglycerols close to 50 %. Also, the use of an inert atmosphere was necessary to avoid over oxidation [34]. The reactions carried out with RP47 and RP48 gave very low conversions as corresponds to their smaller active surfaces, since they were prepared at 700 ºC and 850 ºC respectively (RP47: 8 h, 5 % conversion,

75 % linear diglicerol, 25 % cyclic, RP48: 8 h, 2 % conversion, 50 % cyclic, 50 % linear) [20, 23, 31].

**4. Lipases immobilised on materials prepared with agriresidues derived from rice production.** 

The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/Glycerol = 50 [32]. The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 ºC), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to

over oxidation and triglycerols [33].

diglycerols, 23 min: triglycerols).

rate of 5 °Cmin<sup>−</sup><sup>1</sup>

S-4700 type I, Japan). (Figure11).

The thermal stabilities of the residues in air were analysed by thermal techniques (TGA–DTA) on a Netzsch 409 EP Simultaneous Thermal Analysis device. Approximately 20–30 mg of powdered samples were heated in an air stream of 75 mLmin−1 at a heating rate of 5 °Cmin−1 from ambient to *ca.* 1000°C, using α-alumina as a reference. The termal data were used to design the controlled thermal treatments to produce agriresidue derived materials at temperatures between 500-700 °C, where the organic matter had been decomposed. The microstructure of the agriresidue derived materials was observed by scanning electron microscopy (SEM) (Hitachi S-4700 type I, Japan). (Figure11). character, amount of immobilised enzyme and cost. The use of agroindustrial residues to prepare supports for immobilisation of enzymes can reduce the cost and therefore extend the use of lipases to an industrial scale, since these materaials are cost effective if the technology to make them competitive with commercial ones is developed. In this work the materials used to support enzymes where derived from rice husk (RH) and sunflower (RP) industrial production [13, 35]. The thermal stabilities of the residues in air were analysed by thermal techniques (TGA–DTA) on a Netzsch 409 EP Simultaneous Thermal Analysis device. Approximately 20–30 mg of powdered samples were heated in an air stream of 75 mLmin<sup>−</sup><sup>1</sup> from ambient to *ca.* 1000°C, using α-alumina as a reference. The termal data were used to design the controlled thermal treatments to produce agriresidue derived materials at temperatures between 500-700 ºC, where the organic matter had been decomposed. The microstructure of the agriresidue derived materials was observed by scanning electron microscopy (SEM) (Hitachi

**Figure 11.** SEM images of rice production derived materials. a) RH47 b) RH26

**Figure 11.** SEM images of rice production derived materials. a) RH47 b) RH26

The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/ Glycerol = 50 [32]. The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 °C), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to over oxidation and

The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/Glycerol = 50 [32]. The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 ºC), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to

**Figure 10.** a) Homogeneous reaction of glycerol with NaOH (15 min glycerol, 18-20 minutos cyclic diglycerols). b) Activated RP45 (4 h reaction, 5.7 min: glycolic acid, 7.8 min: glyceric acid, 15 min: glycerol, 18-20 min: cyclic diglycerols, 21 min: linear

**Figure 10.** a) Homogeneous reaction of glycerol with NaOH (15 min glycerol, 18-20 minutos cyclic diglycerols). b) Acti‐ vated RP45 (4 h reaction, 5.7 min: glycolic acid, 7.8 min: glyceric acid, 15 min: glycerol, 18-20 min: cyclic diglycerols, 21

Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material *in situ* to optimise its activity as a basic catalyst. This was done by heating to 500ºC, reaching conversions close to 30 % after 6 h of reaction with selectivity to linear diglycerols close to 50 %. Also, the use of an inert atmosphere was necessary to avoid over oxidation [34]. The reactions carried out with RP47 and RP48 gave very low conversions as corresponds to their smaller active surfaces, since they were prepared at 700 ºC and 850 ºC respectively (RP47: 8 h, 5 % conversion,

Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material *in situ* to optimise its activity as a basic catalyst. This was done by heating to 500°C, reaching conversions close to 30 % after 6 h of reaction with selectivity to linear diglycerols close to 50 %. Also, the use of an inert atmosphere was necessary to avoid over oxidation [34]. The reactions carried out with RP47 and RP48 gave very low conversions as corresponds to their smaller active surfaces, since they were prepared at 700 °C and 850 °C respectively (RP47: 8 h, 5 % conversion, 75 % linear

Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycerides to diglycerides, monoglycerides, fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk preparation, hydrolysing the grease in milk or production of pharmacological substances. In the human body these substances are

diglicerol, 25 % cyclic, RP48: 8 h, 2 % conversion, 50 % cyclic, 50 % linear) [20, 23, 31].

Lipase immobilisation is of interest since it allows their reuse and increases their resistance to inactivation. For industrial applications, several properties are important *i.e.* mechanical strength, chemical and physical stability, hydrophobic/hydrophilic character, amount of immobilised enzyme and cost. The use of agroindustrial residues to prepare supports for immobilisation of enzymes can reduce the cost and therefore extend the use of lipases to an industrial scale, since these materaials are cost effective if the technology to make them competitive with commercial ones is developed. In this work the materials used to support enzymes

Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycer‐ ides to diglycerides, monoglycerides, fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk preparation, hydrolysing the grease in milk or production of pharma‐ cological substances. In the human body these substances are important since they facilitate

**4. Lipases immobilised on materials prepared with agriresidues derived**

The thermal stabilities of the residues in air were analysed by thermal techniques (TGA–DTA) on a Netzsch 409 EP Simultaneous

thermal treatments to produce agriresidue derived materials at temperatures between 500-700 ºC, where the organic matter had been decomposed. The microstructure of the agriresidue derived materials was observed by scanning electron microscopy (SEM) (Hitachi

from ambient to *ca.* 1000°C, using α-alumina as a reference. The termal data were used to design the controlled

**a) b)** 

Thermal Analysis device. Approximately 20–30 mg of powdered samples were heated in an air stream of 75 mLmin<sup>−</sup><sup>1</sup>

75 % linear diglicerol, 25 % cyclic, RP48: 8 h, 2 % conversion, 50 % cyclic, 50 % linear) [20, 23, 31].

**4. Lipases immobilised on materials prepared with agriresidues derived from rice production.** 

where derived from rice husk (RH) and sunflower (RP) industrial production [13, 35].

**Figure 11.** SEM images of rice production derived materials. a) RH47 b) RH26

a) b)

at a heating

triglycerols [33].

196 Agroecology

diglycerols, 23 min: triglycerols).

min: linear diglycerols, 23 min: triglycerols).

important since they facilitate fats adsorption.

**from rice production.**

rate of 5 °Cmin<sup>−</sup><sup>1</sup>

S-4700 type I, Japan). (Figure11).

fats adsorption.

over oxidation and triglycerols [33].

The TXRF analysis of the materials derived from heat treated rice husk (RH47, RH45, RH26) indicated that they contain *ca.* 39 % silicon and 1-2 % calcium and potassium and those derived from sunflower production (RP45 and RP47) 14 % potassium, 12 % calcium, 7 % magnesium, 2 % phosphorous, 1 % iron and 1 % silicon.

The crystallinity of the materials was recorded by X-ray diffraction (XRD) on a Seifert 3000P diffractometer, using Cu Kα1 radiation: *λ* = 0.15406 nm, at 2θ = 5-75 °, with 0.02 ° and 2 sec/ pass (Figure 12). According to these analyses, the RH materials have amorphous structures as correspond to their siliceous nature with small amounts of alkaline and alkaline-earth cations. The materials derived from RP residues were crystalline solids with XRD patterns corre‐ sponding to oxides of potassium, calcium and magnesium when recently calcined, with increasing cristalinities on heat treatment, and their carbonates when in contact with CO2 rich atmosphere (fairchildite (K2Ca(CO3)2) (red lines), calcite (CaCO3) (blue lines)).

The TXRF analysis of the materials derived from heat treated rice husk (RH47, RH45, RH26) indicated that they contain *ca.* 39 % silicon and 1-2 % calcium and potassium and those derived from sunflower production (RP45 and RP47) 14 % potassium, 12 %

The crystallinity of the materials was recorded by X-ray diffraction (XRD) on a Seifert 3000P diffractometer, using Cu Kα<sup>1</sup> radiation: *λ* = 0.15406 nm, at 2θ = 5-75 °, with 0.02 ° and 2 sec/pass (Figure 12). According to these analyses, the RH materials have

when recently calcined, with increasing cristalinities on heat treatment, and their carbonates when in contact with CO2 rich

**Figure 12.** XRD patterns: a) RP47 b) RH26 **Figure 12.** XRD patterns: a) RP47 b) RH26

calcium, 7 % magnesium, 2 % phosphorous, 1 % iron and 1 % silicon.

atmosphere (fairchildite (K2Ca(CO3)2) (red lines), calcite (CaCO3) (blue lines)).

Fourier transformed infrared transmission spectra (FTIR) of materials obtained on a Nicolet 40 FTIR spectrophotometer in the wavenumber range of 4000–400 cm<sup>−</sup><sup>1</sup> , using a 1/100 dilution in KBr indicated the presence of bands at 900-1200 cm-1 and at 400-600 cm-1, corresponding to metal-oxygen bonds (800 cm-1 is O-Si-O symmetric stretching vibrations), given the oxide structure of the materials freshly calcined and bands of OH- at *ca.* 2900-3500 cm-1, that decrease on increasing the treatment temperature due to the loss of water from the OH- groups, carbonate groups are found at 1400-1460 cm-1 and bands close to 2100 cm-1 corresponding to C=O groups present in organic matter, that decrease with the treatment temperature. The specific surface areas measured by N2 adsorption at 77 K after outgassing overnight at 150 °C and employing the BET method for data analyses in a Sorptomatic 1800 instrument (Table 4) indicated that RH derived materials are mesoporous with type IV isotherms and wide pore size distributions, and RP derived materails are non-porous with type II isotherms and non-existent hysteresis loops. Specific surface areas are listed below. Fourier transformed infrared transmission spectra (FTIR) of materials obtained on a Nicolet 40 FTIR spectrophotometer in the wavenumber range of 4000–400 cm−1, using a 1/100 dilution in KBr indicated the presence of bands at 900-1200 cm-1 and at 400-600 cm-1, corresponding to metal-oxygen bonds (800 cm-1 is O-Si-O symmetric stretching vibrations), given the oxide structure of the materials freshly calcined and bands of OH- at *ca.* 2900-3500 cm-1, that decrease on increasing the treatment temperature due to the loss of water from the OH- groups, carbonate groups are found at 1400-1460 cm-1 and bands close to 2100 cm-1 corresponding to C=O groups present in organic matter, that decrease with the treatment temperature.

Material SBET m2 g-1 RH26 63 RH45 98 RH47 16 The specific surface areas measured by N2 adsorption at 77 K after outgassing overnight at 150 °C and employing the BET method for data analyses in a Sorptomatic 1800 instrument (Table 4) indicated that RH derived materials are mesoporous with type IV isotherms and wide pore size distributions, and RP derived materails are non-porous with type II isotherms and nonexistent hysteresis loops. Specific surface areas are listed below.

> RP25 8 RP47 4

mas of 44 was recorded against temperature with a quadrupole mass spectrometer, M3 QMS200 Thermostar coupled to Stanton STA model 781 TG/DTA apparatus. For these analyses approximately 50 mg of the materials were dosed with acetic acid, transferred to **Table 4.** Textural analyses by N2 adsorption desorption and BET calculations

to desorb any loosely bound physically adsorbed acetic acid, until a constant weight was attained. The decomposition of the chemisorbed acetic entities was then achieved by increasing the temperature under a nitrogen flow at a heating rate of 5 °Cmin-1. The amount and temperature of evolution of the CO2 signal gave an indication of the strength and amount of basic sites. The CO2 signal was calibrated from the decomposition of a known amount of calcium oxalate. These measurements indicated the presence of basic groups of high (>500 °C), medium (300-500 °C) and low basicity (100-300 °C) for RP materials, as corresponds to their content in alkaline and alkaline-earth cations and the RH materials had lower amounts of basic centers and of lower strength than the RP materials (Figure 13) [14]. The porosities in pores from 300 μm down to 7.5 nm were determined by mercury intrusion porosimetry (MIP) using CE Instruments Pascal 140/240 porosimeter on samples previously dried overnight at 150 °C, the Washburn equation was employed, assuming a non-intersecting cylindrical pore model and the recommended values for the mercury contact angle and surface tension of 141 ° and 484 mNm−1, respectively. These studies show that materials RH26 and RP47 have pore volumes, mesopores areas, medium pore radii and medium particle sizes as

the crucible placed within the Stanton TG-MS, where they were subsequently flushed with nitrogen gas at room temperature in order

follows: RH26 (0.08 cm3 g-1, 27 m2 g-1, 80 μm and 90 μm), RP47 (0.02 cm3 g-1, 4 m2 g-1, 15 μm and 30 μm).

The TXRF analysis of the materials derived from heat treated rice husk (RH47, RH45, RH26) indicated that they contain *ca.* 39 % silicon and 1-2 % calcium and potassium and those derived from sunflower production (RP45 and RP47) 14 % potassium, 12 %

The crystallinity of the materials was recorded by X-ray diffraction (XRD) on a Seifert 3000P diffractometer, using Cu Kα<sup>1</sup> radiation: *λ* = 0.15406 nm, at 2θ = 5-75 °, with 0.02 ° and 2 sec/pass (Figure 12). According to these analyses, the RH materials have amorphous structures as correspond to their siliceous nature with small amounts of alkaline and alkaline-earth cations. The materials derived from RP residues were crystalline solids with XRD patterns corresponding to oxides of potassium, calcium and magnesium when recently calcined, with increasing cristalinities on heat treatment, and their carbonates when in contact with CO2 rich

Fourier transformed infrared transmission spectra (FTIR) of materials obtained on a Nicolet 40 FTIR spectrophotometer in the

Fourier transformed infrared transmission spectra (FTIR) of materials obtained on a Nicolet 40 FTIR spectrophotometer in the wavenumber range of 4000–400 cm−1, using a 1/100 dilution in KBr indicated the presence of bands at 900-1200 cm-1 and at 400-600 cm-1, corresponding to metal-oxygen bonds (800 cm-1 is O-Si-O symmetric stretching vibrations), given the oxide structure of the materials freshly calcined and bands of OH- at *ca.* 2900-3500 cm-1, that decrease on increasing the treatment temperature due to the loss of water from the OH- groups, carbonate groups are found at 1400-1460 cm-1 and bands close to 2100 cm-1 corresponding to

cm-1, corresponding to metal-oxygen bonds (800 cm-1 is O-Si-O symmetric stretching vibrations), given the oxide structure of the materials freshly calcined and bands of OH- at *ca.* 2900-3500 cm-1, that decrease on increasing the treatment temperature due to the loss of water from the OH- groups, carbonate groups are found at 1400-1460 cm-1 and bands close to 2100 cm-1 corresponding to

The specific surface areas measured by N2 adsorption at 77 K after outgassing overnight at 150 °C and employing the BET method for data analyses in a Sorptomatic 1800 instrument (Table 4) indicated that RH derived materials are mesoporous with type IV isotherms and wide pore size distributions, and RP derived materails are non-porous with type II isotherms and non-existent

> Material SBET m2 g-1

The specific surface areas measured by N2 adsorption at 77 K after outgassing overnight at 150 °C and employing the BET method for data analyses in a Sorptomatic 1800 instrument (Table 4) indicated that RH derived materials are mesoporous with type IV isotherms and wide pore size distributions, and RP derived materails are non-porous with type II isotherms and non-

C=O groups present in organic matter, that decrease with the treatment temperature.

RH26 63 RH45 98 RH47 16 RP25 8 RP47 4

The porosities in pores from 300 μm down to 7.5 nm were determined by mercury intrusion porosimetry (MIP) using CE Instruments Pascal 140/240 porosimeter on samples previously dried overnight at 150 °C, the Washburn equation was employed, assuming a non-intersecting cylindrical pore model and the recommended values for the mercury contact angle and surface tension of 141 ° and

RH26 63 RH45 98 RH47 16 RP25 8 RP47 4

**Material**

g-1, 27 m2

In order to measure the basicity of the solids acetic acid was previously adsorbed onto the powder materials and subsequently the mas of 44 was recorded against temperature with a quadrupole mass spectrometer, M3 QMS200 Thermostar coupled to Stanton STA model 781 TG/DTA apparatus. For these analyses approximately 50 mg of the materials were dosed with acetic acid, transferred to the crucible placed within the Stanton TG-MS, where they were subsequently flushed with nitrogen gas at room temperature in order to desorb any loosely bound physically adsorbed acetic acid, until a constant weight was attained. The decomposition of the chemisorbed acetic entities was then achieved by increasing the temperature under a nitrogen flow at a heating rate of 5 °Cmin-1. The amount and temperature of evolution of the CO2 signal gave an indication of the strength and amount of basic sites. The CO2 signal was calibrated from the decomposition of a known amount of calcium oxalate. These measurements indicated the presence of basic groups of high (>500 °C), medium (300-500 °C) and low basicity (100-300 °C) for RP materials, as corresponds to their content in alkaline and alkaline-earth cations and the RH materials had lower amounts of basic centers and of lower strength than the

The porosities in pores from 300 μm down to 7.5 nm were determined by mercury intrusion porosimetry (MIP) using CE Instruments Pascal 140/240 porosimeter on samples previously dried overnight at 150 °C, the Washburn equation was employed, assuming a non-intersecting cylindrical pore model and the recommended values for the mercury contact angle and surface tension of 141 ° and 484 mNm−1, respectively. These studies show that materials RH26 and RP47 have pore volumes, mesopores areas, medium pore radii and medium particle sizes as

, respectively. These studies show that materials RH26 and RP47 have pore volumes, mesopores areas, medium pore

**SBET m2 g-1**

g-1, 80 µm and 90 µm), RP47 (0.02 cm3

g-1, 4 m2

g-1, 15 µm and

standard.

triglyceride hydrolysis.

**Figure 14.** Trilaurin (a) and triolein (b) formulas.

the mobil phase until only methanol is passed.

**a) b)** 

, using a 1/100 dilution in KBr indicated the presence of bands at 900-1200 cm-1 and at 400-600

calcium, 7 % magnesium, 2 % phosphorous, 1 % iron and 1 % silicon.

**Figure 12.** XRD patterns: a) RP47 b) RH26

**Figure 12.** XRD patterns: a) RP47 b) RH26

hysteresis loops. Specific surface areas are listed below.

radii and medium particle sizes as follows: RH26 (0.08 cm3

wavenumber range of 4000–400 cm<sup>−</sup><sup>1</sup>

198 Agroecology

484 mNm<sup>−</sup><sup>1</sup>

RP materials (Figure 13) [14].

30 µm).

atmosphere (fairchildite (K2Ca(CO3)2) (red lines), calcite (CaCO3) (blue lines)).

C=O groups present in organic matter, that decrease with the treatment temperature.

**Table 4.** Textural analyses by N2 adsorption desorption and BET calculations

existent hysteresis loops. Specific surface areas are listed below.

**Table 4.** Textural analyses by N2 adsorption desorption and BET calculations

In order to measure the basicity of the solids acetic acid was previously adsorbed onto the powder materials and subsequently the mas of 44 was recorded against temperature with a quadrupole mass spectrometer, M3 QMS200 Thermostar coupled to Stanton STA model 781 TG/DTA apparatus. For these analyses approximately 50 mg of the materials were dosed with acetic acid, transferred to the crucible placed within the Stanton TG-MS, where they were subsequently flushed with nitrogen gas at room temperature in order to desorb any loosely bound physically adsorbed acetic acid, until a constant weight was attained. The decomposi‐ tion of the chemisorbed acetic entities was then achieved by increasing the temperature under a nitrogen flow at a heating rate of 5 °Cmin-1.

The amount and temperature of evolution of the CO2 signal gave an indication of the strength and amount of basic sites. The CO2 signal was calibrated from the decomposition of a known amount of calcium oxalate. These measurements indicated the presence of basic groups of high (>500 °C), medium (300-500 °C) and low basicity (100-300 °C) for RP materials, as corresponds to their content in alkaline and alkaline-earth cations and the RH materials had lower amounts of basic centers and of lower strength than the RP materials (Figure 13) [14].

**Figure 13.** TGMS acetic acid decomposition on RP or RH derived materials. **Figure 13.** TGMS acetic acid decomposition on RP or RH derived materials.

The immobilisation process and measurement of activity was undertaken until there was no significant variation in activity. Subsequently, the biocatalyst was filtered, washed, dried over P2O5 and the enzymatic activity was studied at 30 °C, using a Mettler Toledo (modelo DL-50) pH-stato at pH=8.0 with 0.1 N NaOH titrating agent. As reaction medium 19 mL of 1nM tris-HCl buffer at a pH 8.0, 0.6 mL of acetonitrile and 0.4 mL of tripropionine as reaction substrate. A blank test was done to measure spontaneous hydrolisis (without enzyme and only with the triglyceride and the reaction medum). This technique consists of the controlled addition of a basic solution to maintain the pH, being then the titration proportional to the production of acid and therefore to the reaction rate. The enzyme immobilisation on RH materials indicated that during the initial hours the percentage of immobilised enzyme grows but after 24 h there was no more absorption, however, when RP materials were used there was a continuous increment of immobilised enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5, incubated stirring for 1 h at 4 °C and centrifuged to eliminate any solid residue [36]. The immobilisation process and measurement of activity was undertaken until there was no significant variation in activity. Subsequently, the biocatalyst was filtered, washed, dried over P2O5 and the enzymatic activity was studied at 30 °C, using a Mettler Toledo (modelo DL-50) pH-stato at pH=8.0 with 0.1 N NaOH titrating agent. As reaction medium 19 mL of 1nM tris-HCl buffer at a pH 8.0, 0.6 mL of acetonitrile and 0.4 mL of tripropionine as reaction substrate. A blank test was done to measure spontaneous hydrolisis (without enzyme and only with the triglyceride and the reaction medum). This technique consists of the controlled addition of a basic solution to maintain the pH, being then the titration proportional to the production of acid and therefore to the reaction rate.

The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM *p*-nitrophenyl propionate (*p*NPP) as reaction substrate, in kinetic mode, with a wavelength of 405 nm, 30 °C and 2 min. Since the data are given in mU/min, the enzymatic activity was calculated with an extinción coeficient () for the *p*NPP appropiate to The enzyme immobilisation on RH materials indicated that during the initial hours the percentage of immobilised enzyme grows but after 24 h there was no more absorption, however, when RP materials were used there was a continuous increment of immobilised

the wavelength and pH, = 16780 M-1cm-1. The analyses of protein concentration was done by the Bradfor Method using the Biorad reactant and procedure, based on the capacity of dye *Comassie brilliant blue G-250* to change color in the maximum of absorption in the range 465 a 595 nm, according to different concentration of proteins (orange colour becomes blue on the dye bonding to protein at 595 nm. Calibration curves for this procedure were measured with a 50 g/ml solution of bovine serum albumin (BSA) as

The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corresponding fatty acid due to

**a) b)** 

Transesterification reactions were carried out with 50 mM ester concentrations and triglyceride:alcohol molar ratio 1:4, using 2 methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 ºC and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol , water, glacial acetic acid and a dye (*Comassie blue*) (20:80:0.5:0.03) and HPLC composed of a quaternary pump Waters E600, an injector and photodiode detector Varian ProStar and a refractive index detector Waters 2410, with a *Cosmosil* C18 of 4.6 x 150 mm column with a medium particle size of 4.4 m, at 40 °C with a mobil phase of metanol and water acidified with 0.1 %V acetic acid and variable methanol:water ratio. The analysis method is based on time gradient, varying composition and flow of

standard.

triglyceride hydrolysis.

enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5, incubated stirring for 1 h at 4 °C and centrifuged to eliminate any solid residue [36]. **Figure 13.** TGMS acetic acid decomposition on RP or RH derived materials. The immobilisation process and measurement of activity was undertaken until there was no significant variation in activity. Subsequently, the biocatalyst was filtered, washed, dried over P2O5 and the enzymatic activity was studied at 30 °C, using a Mettler Toledo (modelo DL-50) pH-stato at pH=8.0 with 0.1 N NaOH titrating agent. As reaction medium 19 mL of 1nM tris-HCl buffer at a pH 8.0, 0.6 mL of acetonitrile and 0.4 mL of tripropionine as reaction substrate. A blank test was done to measure spontaneous hydrolisis (without enzyme and only with the triglyceride and the reaction medum). This technique consists of the controlled addition of a basic solution to maintain the pH, being then the titration proportional to the production of acid and therefore to the reaction rate.

The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM *p*-nitrophenyl propionate (*p*NPP) as reaction substrate, in kinetic mode, with a wavelength of 405 nm, 30 °C and 2 min. Since the data are given in mU/min, the enzymatic activity was calculated with an extinción coeficient (ε) for the *p*NPP appropiate to the wavelength and pH, ε = 16780 M-1cm-1. The analyses of protein concentration was done by the Bradfor Method using the Biorad reactant and proce‐ dure, based on the capacity of dye *Comassie brilliant blue G-250* to change color in the maximum of absorption in the range 465 a 595 nm, according to different concentration of proteins (orange colour becomes blue on the dye bonding to protein at 595 nm. Calibration curves for this procedure were measured with a 50 μg/ml solution of bovine serum albumin (BSA) as standard. The enzyme immobilisation on RH materials indicated that during the initial hours the percentage of immobilised enzyme grows but after 24 h there was no more absorption, however, when RP materials were used there was a continuous increment of immobilised enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5, incubated stirring for 1 h at 4 °C and centrifuged to eliminate any solid residue [36]. The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM *p*-nitrophenyl propionate (*p*NPP) as reaction substrate, in kinetic mode, with a wavelength of 405 nm, 30 °C and 2 min. Since the data are given in mU/min, the enzymatic activity was calculated with an extinción coeficient () for the *p*NPP appropiate to the wavelength and pH, = 16780 M-1cm-1. The analyses of protein concentration was done by the Bradfor Method using the Biorad reactant and procedure, based on the capacity of dye *Comassie brilliant blue G-250* to change color in the maximum of absorption in the range 465 a 595 nm, according to different concentration of proteins (orange colour becomes blue on the dye bonding to protein

The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corre‐ sponding fatty acid due to triglyceride hydrolysis. at 595 nm. Calibration curves for this procedure were measured with a 50 g/ml solution of bovine serum albumin (BSA) as The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corresponding fatty acid due to

Transesterification reactions were carried out with 50 mM ester concentrations and triglyceride:alcohol molar ratio 1:4, using 2- **Figure 14.** Trilaurin (a) and triolein (b) formulas.

**Figure 14.** Trilaurin (a) and triolein (b) formulas.

methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 ºC and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol , water, glacial acetic acid and a dye (*Comassie blue*) (20:80:0.5:0.03) and HPLC composed of a quaternary pump Waters E600, an injector and photodiode detector Varian ProStar and a refractive index detector Waters 2410, with a *Cosmosil* C18 of 4.6 x 150 mm column with a medium particle size of 4.4 m, at 40 °C with a mobil phase of metanol and water acidified with 0.1 %V acetic acid and variable methanol:water ratio. The analysis method is based on time gradient, varying composition and flow of the mobil phase until only methanol is passed. Transesterification reactions were carried out with 50 mM ester concentrations and triglycer‐ ide:alcohol molar ratio 1:4, using 2-methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 °C and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol, water, glacial acetic acid and a dye (*Comassie blue*) (20:80:0.5:0.03) and HPLC composed of a quaternary pump Waters E600, an injector and photodiode detector Varian ProStar and a refractive index detector Waters 2410, with a *Cosmosil* C18 of 4.6 x 150 mm column with a medium particle size of 4.4 μm, at 40 °C with a

mobil phase of metanol and water acidified with 0.1 %V acetic acid and variable methanol:wa‐ ter ratio. The analysis method is based on time gradient, varying composition and flow of the mobil phase until only methanol is passed.

enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5, incubated stirring for 1 h at 4 °C and

The immobilisation process and measurement of activity was undertaken until there was no significant variation in activity. Subsequently, the biocatalyst was filtered, washed, dried over P2O5 and the enzymatic activity was studied at 30 °C, using a Mettler Toledo (modelo DL-50) pH-stato at pH=8.0 with 0.1 N NaOH titrating agent. As reaction medium 19 mL of 1nM tris-HCl buffer at a pH 8.0, 0.6 mL of acetonitrile and 0.4 mL of tripropionine as reaction substrate. A blank test was done to measure spontaneous hydrolisis (without enzyme and only with the triglyceride and the reaction medum). This technique consists of the controlled addition of a basic solution to maintain the pH, being then the titration proportional to the production of acid and therefore to the reaction rate.

The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM *p*-nitrophenyl propionate (*p*NPP) as reaction substrate, in kinetic mode, with a wavelength of 405 nm, 30 °C and 2 min. Since the data are given in mU/min, the enzymatic activity was calculated with an extinción coeficient (ε) for the *p*NPP appropiate to the wavelength and pH, ε = 16780 M-1cm-1. The analyses of protein concentration was done by the Bradfor Method using the Biorad reactant and proce‐ dure, based on the capacity of dye *Comassie brilliant blue G-250* to change color in the maximum of absorption in the range 465 a 595 nm, according to different concentration of proteins (orange colour becomes blue on the dye bonding to protein at 595 nm. Calibration curves for this procedure were measured with a 50 μg/ml solution of bovine serum albumin (BSA) as

The enzyme immobilisation on RH materials indicated that during the initial hours the percentage of immobilised enzyme grows but after 24 h there was no more absorption, however, when RP materials were used there was a continuous increment of immobilised enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5,

The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corre‐

The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corresponding fatty acid due to

**a) b)** 

Transesterification reactions were carried out with 50 mM ester concentrations and triglyceride:alcohol molar ratio 1:4, using 2 methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 ºC and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol , water, glacial acetic acid and a dye (*Comassie blue*) (20:80:0.5:0.03) and HPLC composed of a quaternary pump Waters E600, an injector and photodiode detector Varian ProStar and a refractive index detector Waters 2410, with a *Cosmosil* C18 of 4.6 x 150 mm column with a medium particle size of 4.4 m, at 40 °C with a mobil phase of metanol and water acidified with 0.1 %V acetic acid and variable methanol:water ratio. The analysis method is based on time gradient, varying composition and flow of

Transesterification reactions were carried out with 50 mM ester concentrations and triglycer‐ ide:alcohol molar ratio 1:4, using 2-methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 °C and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol, water, glacial acetic acid and a dye (*Comassie blue*) (20:80:0.5:0.03) and HPLC composed of a quaternary pump Waters E600, an injector and photodiode detector Varian ProStar and a refractive index detector Waters 2410, with a *Cosmosil* C18 of 4.6 x 150 mm column with a medium particle size of 4.4 μm, at 40 °C with a

The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM *p*-nitrophenyl propionate (*p*NPP) as reaction substrate, in kinetic mode, with a wavelength of 405 nm, 30 °C and 2 min. Since the data are given in mU/min, the enzymatic activity was calculated with an extinción coeficient () for the *p*NPP appropiate to the wavelength and pH, = 16780 M-1cm-1. The analyses of protein concentration was done by the Bradfor Method using the Biorad reactant and procedure, based on the capacity of dye *Comassie brilliant blue G-250* to change color in the maximum of absorption in the range 465 a 595 nm, according to different concentration of proteins (orange colour becomes blue on the dye bonding to protein at 595 nm. Calibration curves for this procedure were measured with a 50 g/ml solution of bovine serum albumin (BSA) as

centrifuged to eliminate any solid residue [36].

incubated stirring for 1 h at 4 °C and centrifuged to eliminate any solid residue [36].

**Figure 13.** TGMS acetic acid decomposition on RP or RH derived materials.

sponding fatty acid due to triglyceride hydrolysis.

standard.

200 Agroecology

**Figure 14.** Trilaurin (a) and triolein (b) formulas.

**Figure 14.** Trilaurin (a) and triolein (b) formulas.

the mobil phase until only methanol is passed.

standard.

triglyceride hydrolysis.

Transesterification of triglycerides with methanol and ethanol produce mainly methyl and ethyl oleate and secondary compounds such as fatty acid esters (mono- and dilaurin, mono‐ olein and diolein) and the corresponding fatty acid due to the triglyceride hydrolysis. On using Novozym 435, trilaurin disappears after 24 h of reaction, because when the trilaurin is consumed, ethanol interacts with the diglyceride. Regarding the use of Lipozyme TL-IM, ethyl laureate formation is slower, although trilaurin also gets consumed and the formation of acid is lower, because Lipozyme TL-IM has a lower amount of water than Novozym 435. In the case of Lipozyme RM-IM, the formation of ethyl laureate is not so important as in the cases stated before, and at 24 h there is still a lot of unreacted trilaurin.


**Table 5.** Textural and reactivity data for enzyme immobilised on agriresidue derived materials.

Amongst the materials used, those derived from rice husk show higher capacities for lipase absorption. It should be noted that RH45 and RH26 are the materials that have highest surface areas, but taking into account the amount of enzyme absorbed, it is clear that not only the textural characteristics are defining the behaviour of the supports but the amount and strength of the basic sites was also important, much higher in the RP materials. RH26 was chosen for preparation of immobilised enzyme for biodiesel synthesis in comparison with commercial enzymes. The ROL enzyme supported on RH26 prepared in the group had a higher activity than Novozyme 435 and similar to that of Lipozyme TL-IM, both lipases widely used in biocatalysis. Trilaurin was consumed in *ca.* 24 h with ethyl laureate formation close to that of Novozyme 435 and lauric acid due to the hydrolysis produced by the water contained in the enzyme.


**Table 6.** Comparison of reactivity of commercial and agriresidue supported enzymes.

The biocatalysts prepared by immobilisation of the lipase on agriresidue derived materials, given their renewable origin and low cost, seem to be an attractive option for reducing costs and environmental impact of these processes [37, 38].
