**9.1.1 Chemically catalyzed process**

Verhé and coworkers (Verhé *et al.*, 2008) reported a process of converting the deodorizer distillates to biodiesel using methanol in a weight ratio 1:1 and 5 % w/w sulphuric acid as catalyst, at 75 °C for 5 h. Under the mentioned conditions, the FFA have undergone esterification while MAG reacted *via* transesterification, resulting in methyl esters. The crude biodiesel was further washed with 20 % water for 15 min, dried and distilled in order to increase the quality of the methyl esters. The distillation pitch was further processed for the recovery of sterols and tocopherols.

Facioli and Arellano (Facioli & Barrera-Arellano, 2002) described a process to obtain ethyl esters from SODD. SODD contained 47.5 % FFA C18:1, 26.2 % acylglycerols and 26.2 % unsaponifiable matter using concentrated sulphuric acid as catalyst. The optimum conditions found in this study were for EtOH:FFA between 6.4:1 to 11.2:1, H2SO4 from 0.9- 1.5 % and reaction time from 1.3 h to 2.6 h. Under the described conditions a conversion of 94 % of the fatty acids to ethyl esters was achieved. Tocopherols losses were below 5.5 %. A molar excess of ethanol in relation to SODD:FFA was found to be necessary to obtain the best conversion.

Hammond and coworkers (Hammond & Tong, 2005) described a three-stage acid catalyzed esterification using a molar ratio acid oil:methanol:sulphuric acid of 1:1.3:0.03 for the first stage (25 h). The reaction mixture was centrifuged, the supernatant lipid phase was separated from the sludge (glycerol, water, acid and methanol), and further reacted with methanol and acid, keeping the previous mentioned ratios of unreacted lipid:methanol:sulphuric acid.

Extraction and Enzymatic Modification of

by other authors (Hammond & Tong, 2005).

**9.2.1 Enzymatically catalyzed process** 

unesterified fatty acids and MAG.

**9.2.2 Non-catalytic process** 

emulsifier.

catalytic conditions.

**9.2 Production of biodiesel via acylglycerols route** 

Functional Lipids from Soybean Oil Deodorizer Distillate 471

Du and coworkers (Du *et al.*, 2007) investigated the enzymatic esterification of SODD containing 28 % FFA, 60 % TAG and 6 % tocopherols. The reaction was lipase mediated methanolysis using Novozym 435 as catalyst, at 40 °C in a solvent free medium. The enzyme kept its activity after being reused for 10 cycles, each cycle of 24 h. The highest biodiesel yield of 95 % was achieved by adding 10 fold of 3 Å molecular sieves (based on the maximum water produced from FFA esterification). The investigation of the lipase to methanol tolerance revealed that the lipase could maintain its stability and activity in the presence of even 3 molar concentration of methanol. This tolerance was attributed to the presence of other compounds apart from triglycerides, namely FFA, sterols and tocopherols. A linear relationship between the FFA content and the lipase tolerance to methanol was observed but the presence of sterols and tocopherols showed no effect. The correlation between the initial FFA present in the feedstock and the rate of conversion was confirmed

Another approach reported in the literature consists on esterification of FFA with glycerol to form acylglycerols, followed by conventional transesterification. Synthesis of MAG from deodorizer distillate was mainly studied due to the large number of applications as additives (e.g. enhancing plasticity of fats) in the food, medicine and cosmetic industry. Among synthesized acylglycerols, the monoester has the highest surface activity and therefore, its concentration is very important for direct utilization of the reaction mixture as

Although the use of a large number of different heterogeneous catalysts have been reported in literature, most of the research has been done on the synthetic samples and less on the side stream refining products. Different studies summarized hereunder describe processes for synthesis of acylglycerols as an intermediate step in the production of biodiesel/biofuels. These processes are catalyzed either chemically or enzymatically, or conducted under non-

Tangkam and coworkers (Tangkam *et al.*, 2008) described the enzymatic esterification in a solvent free medium of different deodorizer distillates resulting from the refining of various vegetable oils. A direct esterification of mixed distillates (61 % FFA and 39 % acylglycerols) with glycerol using immobilized lipase B from *Candida Antarctica* (Novozym 435) led to moderate proportions (46 %) of DAG. Application of a two-stage reaction consisting of a hydrolysis step of deodorizer distillate to increase the FFA content followed by esterification with glycerol led to a higher formation (>61 %) of DAG. Furthermore, it was observed that the high initial concentration of free fatty acids in the distillate has a positive influence on the concentration of DAG in the final product (>71 %). This observation is consistent with other literature data (Yamada *et al.*, 1999). Enrichment of DAG in the final products by shortpath vacuum distillation led to concentrates containing up to 94 % DAG, ~ 5 % TAG and no

Smet (Smet, 2008) described a process for the esterification of fatty acid distillate (93 % FFA) with technical grade glycerol. The reaction was carried out in a high pressure Parr reactor

It was seen that the reaction proceeded rapidly during the first hour of reaction and then slowed down considerably. In contrast, the second and third stage showed a gradual increase in FAME over time. The maximum FAME conversion obtained for 12 tested acid oils averaged 81%. However, the ester phase could not be increased above 85% even after a fourth-stage reaction or if a base catalyst (sodium methoxide) was used in large excess. If higher amount of methanol was used, the initial reaction tended to go faster, but the reaction reached the plateau in a short time. Furthermore, an increase in the acid catalyst concentration above 1.2 % did not affect the initial reaction rate.

#### **9.1.2 Enzymatically catalyzed process**

Several enzymatic methods have been developed for the conversion of fatty acids into FAMEs or FAEEs with positive results. One of the main disadvantages of use of biocatalysts is the high price compared to chemical catalyst, although unfortunately, no rigorous economical viability of these enzymatic procedures has been reported.

Facioli and Arellano (Facioli & Barrera-Arellano, 2001) investigated the enzymatic esterification of the free fatty acids from SODD with ethanol using immobilized fungal lipase (Lipozyme IM) as biocatalyst. SODD contained 47.5 % FFA, 26.22 % neutral oil and 26.23 % unsaponifiable matter. The effect of three independent variables: temperature, enzyme concentration and EtOH:FFA molar ratio on the conversion rate of FFA to ethyl esters was studied. The best conversion (above 88 %) was obtained with EtOH:FFA ratio 1.7- 3.2:1, temperature in the range 46.4 °C to 53.6 °C, lipase concentration from 10.7 to 23.0% and the reaction time of 2 h. All three variables had statistically significant effect on the conversion of the FFAs to ethyl esters. During the above mentioned esterification process no tocopherols losses were observed.

The esterification of SODD with butanol using *Mucor miehei* lipase as a biocatalyst and supercritical carbon dioxide (SC-CO2) has been described by Nagesha and coworkers (Nagesha, 2004). The SODD contained 56.0 % neutral oil, 25.3 % FFA, 7.2 % sterols, 2.9 % tocopherols, 0.6 % hydrocarbons and 0.13 % moisture. It was preliminary filtered in order to remove sediments and sterols and enzymatic hydrolyzed to free fatty acid using immobilized lipase (*Candida rugosa*) in SC-CO2 reactor unit. The operational conditions were as follows: pressure 160 bar, temperature 45 °C, moisture content 60 % (w/w) and enzyme concentration 200 U/g of SODD. Hydrolyzed SODD containing 87.8 % (w/w) FFA was further esterified for 3h in presence of butanol (1.2 M) using 15 % enzyme (w/w) (*M. miehei*), pressure 120 bar and temperature 35 °C. The maximum yield of 95 % FABE was achieved.

The high content of residual glycerides (3.10 %) present in the final FABE precluded its direct use as biodiesel. However, the process was designed as preliminary step for the purification of tocopherols, since hydrolysis/esterification helps their recovery.

Wang and coworkers (Wang *et al.*, 2006) described a process for simultaneously conversion of FFA (28 %) and acylglycerols (60 %) from SODD to alkyl esters using a mixture of two enzymes (3 % Lipozyme TL IM and 2 % Novozym 435) in the presence of *tert*-butanol as cosolvent. It was found that the negative effects on the enzyme stability caused by the excessive methanol ratio and by-product glycerol could be minimized by using *tert*-butanol. The lipase activity remained stable after 120 cycles. The maximum yield of FAME (84 %) was achieved with an increase of *tert*-butanol content up to 80 % (based on the oil weight). However, a further increase of the solvent resulted in a decrease of the FAME yield which was explained by the dilution effect on reactants.

It was seen that the reaction proceeded rapidly during the first hour of reaction and then slowed down considerably. In contrast, the second and third stage showed a gradual increase in FAME over time. The maximum FAME conversion obtained for 12 tested acid oils averaged 81%. However, the ester phase could not be increased above 85% even after a fourth-stage reaction or if a base catalyst (sodium methoxide) was used in large excess. If higher amount of methanol was used, the initial reaction tended to go faster, but the reaction reached the plateau in a short time. Furthermore, an increase in the acid catalyst

Several enzymatic methods have been developed for the conversion of fatty acids into FAMEs or FAEEs with positive results. One of the main disadvantages of use of biocatalysts is the high price compared to chemical catalyst, although unfortunately, no rigorous

Facioli and Arellano (Facioli & Barrera-Arellano, 2001) investigated the enzymatic esterification of the free fatty acids from SODD with ethanol using immobilized fungal lipase (Lipozyme IM) as biocatalyst. SODD contained 47.5 % FFA, 26.22 % neutral oil and 26.23 % unsaponifiable matter. The effect of three independent variables: temperature, enzyme concentration and EtOH:FFA molar ratio on the conversion rate of FFA to ethyl esters was studied. The best conversion (above 88 %) was obtained with EtOH:FFA ratio 1.7- 3.2:1, temperature in the range 46.4 °C to 53.6 °C, lipase concentration from 10.7 to 23.0% and the reaction time of 2 h. All three variables had statistically significant effect on the conversion of the FFAs to ethyl esters. During the above mentioned esterification process no

The esterification of SODD with butanol using *Mucor miehei* lipase as a biocatalyst and supercritical carbon dioxide (SC-CO2) has been described by Nagesha and coworkers (Nagesha, 2004). The SODD contained 56.0 % neutral oil, 25.3 % FFA, 7.2 % sterols, 2.9 % tocopherols, 0.6 % hydrocarbons and 0.13 % moisture. It was preliminary filtered in order to remove sediments and sterols and enzymatic hydrolyzed to free fatty acid using immobilized lipase (*Candida rugosa*) in SC-CO2 reactor unit. The operational conditions were as follows: pressure 160 bar, temperature 45 °C, moisture content 60 % (w/w) and enzyme concentration 200 U/g of SODD. Hydrolyzed SODD containing 87.8 % (w/w) FFA was further esterified for 3h in presence of butanol (1.2 M) using 15 % enzyme (w/w) (*M. miehei*), pressure 120 bar and temperature 35 °C. The maximum yield of 95 % FABE was achieved. The high content of residual glycerides (3.10 %) present in the final FABE precluded its direct use as biodiesel. However, the process was designed as preliminary step for the

purification of tocopherols, since hydrolysis/esterification helps their recovery.

Wang and coworkers (Wang *et al.*, 2006) described a process for simultaneously conversion of FFA (28 %) and acylglycerols (60 %) from SODD to alkyl esters using a mixture of two enzymes (3 % Lipozyme TL IM and 2 % Novozym 435) in the presence of *tert*-butanol as cosolvent. It was found that the negative effects on the enzyme stability caused by the excessive methanol ratio and by-product glycerol could be minimized by using *tert*-butanol. The lipase activity remained stable after 120 cycles. The maximum yield of FAME (84 %) was achieved with an increase of *tert*-butanol content up to 80 % (based on the oil weight). However, a further increase of the solvent resulted in a decrease of the FAME yield which

concentration above 1.2 % did not affect the initial reaction rate.

economical viability of these enzymatic procedures has been reported.

**9.1.2 Enzymatically catalyzed process** 

tocopherols losses were observed.

was explained by the dilution effect on reactants.

Du and coworkers (Du *et al.*, 2007) investigated the enzymatic esterification of SODD containing 28 % FFA, 60 % TAG and 6 % tocopherols. The reaction was lipase mediated methanolysis using Novozym 435 as catalyst, at 40 °C in a solvent free medium. The enzyme kept its activity after being reused for 10 cycles, each cycle of 24 h. The highest biodiesel yield of 95 % was achieved by adding 10 fold of 3 Å molecular sieves (based on the maximum water produced from FFA esterification). The investigation of the lipase to methanol tolerance revealed that the lipase could maintain its stability and activity in the presence of even 3 molar concentration of methanol. This tolerance was attributed to the presence of other compounds apart from triglycerides, namely FFA, sterols and tocopherols. A linear relationship between the FFA content and the lipase tolerance to methanol was observed but the presence of sterols and tocopherols showed no effect. The correlation between the initial FFA present in the feedstock and the rate of conversion was confirmed by other authors (Hammond & Tong, 2005).
