Development of biodiesel: Current scenario

Abstract Fuels are inevitable for industrial development and growth of any country. The life span of fossil fuel resources has always been terrifying. Biodiesel, a renewable source of energy seems to be an ideal solution for global energy demands including India as well. The current review is addressed to various aspects of biodiesel production. Latest literature has been critically reviewed and consulted. 5. Variables affecting the transesterification reaction Transesterification reaction is quite sensitive to various parameters. The reaction is either incomplete or the yield is reduced to a significant extent if the parameters are not optimised. These parameters include free fatty acids (FFAs), water content, molar ratio of alcohol to oil, catalyst, reaction temperature and stirring. Each parameter is equally important to achieve a high quality biodiesel which meets the regulatory standards. 5.1. Free fatty acids Free fatty acids (FFAs) are the saturated or unsaturated monocarboxylic acids that occur naturally in fats, oils or greases but are not attached to glycerol backbones [22]. Higher amount of free fatty acids leads to higher acid value. Vegetable oils should have free fatty acids within a desired limit for alkaline transesterification, beyond which either the reaction will not take place or the yield will be too less. Table 3 [7,18,23 27] depicts the level of FFA worked out by researchers. It is clear from the table that the FFA level in the oil should be below a desired level (ranging from less than 0.5% to less than 3%) for alkaline transesterification to take place. The application of the acid catalyst is to reduce the free fatty acids to a level safe enough for alkali transesterification. Else, the product formed will be soap instead of esters. During acid catalysed process, the FFA react with alcohol to produce esters but simultaneously, water is also produced which inhibits the transesterification reaction [28]. An alkali catalyst proceeds at around 4000 times faster than with the same amount of acid catalyst and hence is preferred over the acid catalyst after the acid value is reduced to the desired limit [29]. Hence, the transesterification reaction is one-step process for oils with FFA within the range and is a two-step process for oils with FFA exceeding the range. In a two-step process, acid esterification is followed by alkali transesterification.

5.2. Water content The raw materials being used as starting material should be free from water content. Water content as low as 0.1% has been reported to decrease the conversion of ester to a significant extent [28]. Srivastava and Verma [30] reported the removal of the moisture content by heating the oil in an oven for 1 h at 110C before starting the transesterification reaction. Meher et al. [31] dissolved the catalyst, potassium hydroxide in methanol just before starting the transesterification reaction in an attempt to prevent the moisture absorbance. 5.3. Alcohol and molar ratio employed

Lower alcohols such as methanol, ethanol, propanol, etc. can be employed for transesterification reaction without any significant difference in the yield of the product. Methanol is toxic but is preferred among the others owing to its low cost. Ethanol is not preferred because of its low reactivity compared to methanol. Viscosity of ethyl esters is slightly higher and low temperature properties (cloud point, pour point) are slightly lower than those of methyl esters [32]. These findings make methanol more amenable for use as alcohol in biodiesel development. However, methanol has a lower boiling point of 64.7C and the transesterification reaction is carried out at a temperature which is near to this temperature. Vapours of methanol, which are highly toxic and can cause permanent blindness, are likely to be present near the site of reaction set up. Hence, appropriate measures have to be ensured for the safety of personnel working around. A new technique has been developed by the researchers [33] where alcohol in supercritical condition is used for the completion of transesterification reaction in shorter period of time without using any catalyst. The reason for the shorter time span is that the oil and supercritical alcohol exist in the same phase. In the supercritical transesterification method, a conversion of 5095% was achieved in first 10 min. Presence of water was a source of interference in catalytic transesterification, whereas, the presence of water had shown a positive affect in methyl ester conversion by supercritical method [32]. The commonly employed molar ratio for two-step transesterification is 6:1 for acid transesterification and 9:1 for alkali catalyzed transesterification. However, the optimum molar ratio has shown to differ a little depending on the raw oil taken and its acid value. For single step transesterification reaction, 10:1 molar ratio has been used more often, although an optimum molar ratio varying from 6:1 to 13:1 has been employed by other researchers [34]. A molar ratio higher than the optimum value reduces the yield and makes the separation process of esters and glycerol difficult. High molar (40:1) is needed where supercritical methanol is used for transesterification method [33]. 5.4. Types and amount of catalysts A catalyst is needed to improve the transesterification reaction and yield [35]. Homogeneous catalyst has been in use at present at industrial level for production of biodiesel. Sulphuric acid (H2SO4) is the commonly used catalyst during acid transesterification whereas sodium hydroxide (NaOH) and potassium hydroxide (KOH) are the catalyst used for alkaline transesterification [34]. Sodium methoxide (CH3ONa) and potassium methoxide (CH3OK) are the other homogeneous catalyst which perform better than NaOH and KOH in terms of yield. Formation of small amount of water during the transesterification reaction results in lower yield of biodiesel with NaOH or KOH as a catalyst whereas CH3ONa or CH3OK show higher yield because there is no water formation as by-product during the reaction [36]. Among KOH and NaOH, Vicente et al. [37] reported higher yield with KOH (91.67%) as compared to that of NaOH (85.9%). However, the purity of the ester reported was similar with these two catalysts. Contrary to this, Leung and Guo [36] have reported different amounts of catalysts required to achieve the same conversion of methyl esters. The amounts of catalysts required were 1.1, 1.3 and 1.5 wt.% for NaOH, CH3ONa and KOH, respectively to get maximum conversion of methyl esters. This was attributed to the smallest molar mass of NaOH (40 g/mol) as compared to CH3ONa (54 g/mol) and KOH (56 g/mol). The amount of acid catalyst used to reduce the acid value ranges from 0.65% to 1.43% (v/v) H2SO4. However, the amount most commonly used by researchers is 1% (v/v) of 100% H2SO4. Optimum amount of H2SO4 as catalyst only is to be added, as higher volume may burn the oil and can darken the product. The amount of NaOH or KOH used during alkali catalyzed transesterification reaction ranges from 0.7% to 1.5% by weight, depending on the nature of oil [34]. Lower amount of catalyst results in incomplete reaction, whereas higher amount of catalyst causes soap formation. Hence, an optimum amount of catalyst is a very important parameter in the transesterification reaction. Homogeneous catalysts have few disadvantages in their application. These catalysts have to be removed from the final product with repeated washing with distilled water, which give rise to colossal generation of wastewater [38]. 5.5. Reaction temperature As a general rule, transesterification reaction is tried to be accomplished at lowest possible temperature and time [44]. The commonly employed temperature ranges from as low as room temperature to up to

65C. Transesterification reaction has been reported to be influenced positively with increase in temperature. The boiling point of methanol is 64.7C and hence the transesterification reaction is carried out within this range as a temperature higher than this may burn methanol. Higher temperature also favours saponification and hence must be avoided [18]. Temperature of 350C has been considered to be optimum while using supercritical methanol [45]. 5.6. Rate and mode of stirring Scientific workers have reported stirring to be an equally important parameter for synthesis of biodiesel. Transesterification reaction was incomplete with 180 rotations per minute (rpm) of stirring, while the yield was same with 360 rpm and 600 rpm [31]. Mixing of reactants at 400 rpm with magnetic stirrer was performed by Veljkovic et al. [46] to get the optimum yield. A higher rate of stirring of 1100 rpm has also been reported to achieve maximum yield of biodiesel. Not much information on mode of stirring is available. However, a better yield with mechanical stirring has been reported than with magnetic mode of stirring [10,36]. 5.7. Purification of the final product The methyl esters obtained after one-step transesterification reaction were treated with hot water at 70C (1/5 of methyl ester volume) and 5% H3PO4 (aq) at 50C. After discharge of the wastewater, the ester layer was dried in vacuum and checked with ceric ammonium nitrate reagent for glycerol. Similar process was adopted for two-step transesterification method [19]. The fatty acid methyl esters were distilled by Wang et al. [47] at 180C under vacuum (40 5 mmHg) and collected. The distillation was assumed to be completed when the temperature reached 240C (40 5 mmHg) and the final yield obtained was 93.0 wt.%. Biodiesel separated after acidic transesterification was washed with petroleum ether and then with hot water (50C) until the washing reached a neutral pH. Srivastava and Verma [30] washed the biodiesel with 10% H3PO4 by bubble wash method after separation from glycerol. Biodiesel was further purified by passing air by aquarium stone for at least 24 h. The product biodiesel is also treated by washing it with hot distilled water to remove the dissolved impurities such as catalysts, alcohol, etc. Silica gel has been reported to be used for removing the catalyst from the biodiesel product [48]. The yield of biodiesel is calculated as follows [36]:

Conversion of triglyceride to alkyl ester is calculated by titration method [40], i.e. by measurement of acid value:

PCR Amplification of 18S Rrna, Single Cell Protein Production and Fatty Acid Evaluation of Some Naturally Isolated Microalgae
Abstract Microalgae were isolated during a screening program from soil samples collected from paddy-fields of Fars province, south of Iran. The protein content was assayed by the Kochert method. Total genomic DNA were isolated and used for PCR amplification of the 18S rRNA gene. The sequences were determined for 12 species of microalgae. Some bioinformatic tools were used for more investigation on these biologic data. Total lipids from five microalgal species were extracted and used for determination of different types of fatty acids by gas chromatographymass spectrometry method. In our experiments the green algae yielded a maximum protein of about 42% 1.64. The DNA

sequences were published in the NCBI under specific accession numbers. The composition of fatty acids was mainly, myristic acid, palmitic acid, oleic acid, a-linolenic acid, and c-linolenic acid. 2.5. Esterification of fatty acids Extracted crude lipids of microalgae (0.5 g) were dissolved in 3 mL of methanol in the presence of catalytic amount of sulphuric acid. The mixture was heated to reflux using DienStark apparatus. After cooling, the reaction mixture was washed twice with 4 mL of saturated sodium hydrogen carbonate aqueous solution, dried over anhydrous sodium sulphate and the solvent was removed by distillation under reduced pressure to give an oily substance. Then the thin-layer chromatography (TLC) was applied to monitor the progress of methyl esterification on the extracted fatty acids. The TLC was performed using silica gel 60 F254 plates (20 _ 20 cm, 250 lm layer thickness, Merck, Darmstadt, Germany) and pure chloroform as solvent system. The obtained oily substance was injected in GC/MS for analysis. 3.3. Fatty acid identification Fatty acids in the five strains of microalgae (Chlorella vulgaris MCCS 013, D. salina MCCS 001, D. salina CCAP 19/18, Scenedesmus obliquus strain 019, Scenedesmus rubescens MCCS 018) were primarily esterified and then identified through GC/MS analysis. The GC/MS analyses were carried out on the product of esterification (fatty acids (FAs)/fatty acid methyl esters (FAMEs)) in the above mentioned microalgae strains. The total fatty acid content of each strain was about 25% of dry weight of biomass. The identification of FAs/FAMEs was performed through the comparison of their mass spectra with those in Wiley libraries. As shown in these tables (Tables 26), different types of fatty acids are detected in the five sample strains. Esterification of the studied microalgal fatty acids is the choice to identify those using GC/MS (Butte, 1983; Mller, Husamann, & Nalik, 1990). GC/MS analysis allowed us to discover several types of fatty acids, such as chained, branched, monounsaturated, polyunsaturated, alkynoic and dioic fatty acids in some naturally isolated microalgal strains and showed the presence of at least 50 fatty acids in them. GC/MS analysis of fatty acids requires the conversion of fatty acids to fatty acid methyl esters, which is often done by saponification and esterification with methanol (Mller et al., 1990). The major fatty acids in our five studied strains of microalgae were shown to be undecanoic acid, myristic acid, palmitic acid, and eicosanoic acid (Tables 26). The shortest identified fatty acids was propenoic acid with 3 carbon atoms in D. salina MCCS 001 and the longest was octatriacontanoic acid with 38 carbon atoms in D. salina MCCS 001 (Table 3). Furthermore D. salina MCCS 001 showed the most diverse strain in the studied microalgae which had 34 types of fatty acids. Undecanoic acid was found in all of the studied strains but some fatty acids were found only in one strain such as 2-propenoic acid and pentanoic acid, in D. salina MCCS 001 (Table 3), nonanoic acid, 7-hexadecenoic acid, 9,15-octadecadienoic acid, 10,13-octadecadienoic acid and 12,15octadecadienoic acid in S. obliquus strain 019 (Table 6), 9,12-hexadecadienoic acid and 9-octadecenoic acid in S. rubescens (Table 5), 7,10,13-hexadecatrienoic acid, 11,14,17-eicosatrienoic acid, and 9,12,15octadecatrienoic acid (a-linolenic acid) in C. Vulgaris MCCS 013 (Table 2). Furthermore, important fatty acids like oleic acid, a monounsaturated (x9) fatty acid in S. rubescens MCCS 018 (Table 5), 6,9,12octadecatrienoic acid (c-linolenic acid), a polyunsaturated (x6) fatty acid in D. salina CCAP 19/18 (Table 4) and S. rubescens MCCS 018 (Table 5), 7,10,13-hexadecatrienoic acid, a polyunsaturated (x3) fatty acid in C. vulgaris MCCS 013 (Table 2), 11,14,17-eicosatrienoic acid, a polyunsaturated (x3) fatty acid in C. vulgaris MCCS 013 (Table 2) and 9,12,15-octadecatrienoic acid (a-linolenic acid), a polyunsaturated (x3) fatty acid in C. vulgaris MCCS 013 (Table 2) were also identified.

Study of Increasing Lipid Production from Fresh Water Microalgae Chlorella Vulgaris
Study of increasing lipid production from fresh water microalgae Chlorella vulgaris was conducted by investigating several important factors such as the effect of CO2 concentration, nitrogen depletion and harvesting time as well as the method of extraction. The drying temperature during lipid extraction from

algal biomass was found to affect not only the lipid composition but also lipid content. Drying at very low temperature under vacuum gave the best result but drying at 60C still retained the composition of lipid while total lipid content decreased only slightly. Drying at higher temperature decreased the content of triacylglyceride (TG). As long as enough pulverization was applied to dried algal sample, ultrasonication gave no effect whether on lipid content or on extraction time. In addition to the increase of total lipid content in microalgal cells as a result of cultivating in nitrogen depletion media, it was found that changing from normal nutrient to nitrogen depletion media will gradually change the lipid composition from free fatty acid-rich lipid to lipid mostly contained TG. Since higher lipid content was obtained when the growth was very slow due to nitrogen starvation, compromising between lipid content and harvesting time should be taken in order to obtain higher values of both the lipid content and lipid productivity. As the growth was much enhanced by increasing CO2 concentration, CO2 concentration played an important role in the increase of lipid productivity. At low until moderate CO2 concentration, the highest lipid productivity could be obtained during N depletion which could surpassed the productivity during normal nutrition. At high CO2 concentration, harvesting at the end of linear phase during normal nutrition gave the highest lipid productivity. However, by reducing the incubation time of N depletion, higher lipid content as well as higher lipid productivity may still be achieved under this condition. 2.3. Lipid extraction Dry extraction procedure according to Zhu et al. (2002) as a modification of the wet extraction method by Bligh and Dyer (1959) was used to extract the lipid in microalgal cells. Typically, cells were harvested by centrifugation at 8500 rpm for 5 min and washed once with distilled water. After drying the samples using freeze drier, the samples were pulverized in a mortar and extracted using mixture of chloroform:methanol (2:1, v/v). About 50 mL of solvents were used for every gram of dried sample in each extraction step. After stirring the sample using magnetic stirrer bar for 5 h and ultrasonicated for 30 min, the samples were centrifuged at 3000 rpm for 10 min. The solid phase was separated carefully using filter paper (Advantec filter paper, no. 1, Japan) in which two pieces of filter papers were applied twice to provide complete separation. The solvent phase was evaporated in a rotary evaporator under vacuum at 60 8C. The procedure was repeated three times until the entire lipid was extracted. The effect of solvents having different polarities for extracting the lipid, as well as the effect of drying temperature and ultrasonication time were investigated in this study. 3.1.2. Effect of drying temperature Fig. 1 shows the effect of drying temperature on the lipid content. Heating at 60C resulted in a slight decrease of lipid content but when heating was conducted at 80C or higher temperature, the lipid content decreased significantly. Table 1 shows that the content of TG tend to decrease when higher temperature was applied for the drying of algal sample. Oxidation of fatty acid upon exposing to high temperature has been reported (Oehrl et al., 2001). They reported that unsaturated fatty acid, especially polyunsaturated free fatty acid (PUFA), was more susceptible to oxidation than saturated fatty acid. The reasonable explanation of the degradation of TG in Table 1 was the oxidation of TG at high temperature for 12 h. The TG in microalgal cells obtained in the present works should consist of highly unsaturated fatty acids. Bockisch (1998) and Choe and Min (2007) also reported that the degradation of TG by oxidation also resulted in the formation of hydroperoxide group (OOH) in the chain. The hydroperoxides formed can react further to aldehydes, ketones, and fatty acids. The results in Table 1 in which the content of other compounds was increased seemed to confirm the previous observation. The others in Table 1 should therefore contained aldehydes and ketones. The resistance of DG after drying may indicate that they consisted of saturated fatty acids which were not easily broken down in comparison to the highly unsaturated fatty acid that may construct the TG.

BIODIESEL PRODUCTION: A REVIEW

SYNTHESIS OF BIODIESEL VIA ACID CATALYSIS

Edgar Lotero, Yijun Liu, Dora E. Lopez, Kaewta Suwannakarn, David A. Bruce, and James G. Goodwin, Jr. Soap forms when the metal hydroxide catalyst reacts with FFAs in the feedstock (Figure 3a). Soap production gives rise to the formation of gels, increases viscosity, and greatly increases product separation cost.2 The alcohol and catalyst must also comply with rigorous specifications. The alcohol as well as the catalyst must be essentially anhydrous (total water content must be 0.1-0.3 wt % or less).3 This is required since it is assumed that the presence of water in the feedstock promotes hydrolysis of the alkyl esters to FFAs (Figure 3b) and, consequently, soap formation.

The fact that the homogeneous acid-catalyzed reaction is about 4000 times slower than the homogeneous base-catalyzed reaction has been one of the main reasons. However, acid-catalyzed transesterifications hold an important advantage with respect to base-catalyzed ones: the performance of the acid catalyst is not strongly affected by the presence of FFAs in the feedstock. In fact, acid catalysts can simultaneously catalyze both esterification and transesterification. A simplified block flow diagram (BFD) of a typical acid-catalyzed process is shown in Figure 4 illustrating the most important steps in biodiesel production.

Myristic acid IUPAC name: tetradecanoic acid Molar mass: 228.37092 Molecular formula: C14H28O2

Linoleic acid IUPAC name: all-cis-9,12-octadecadienoic acid Molar mass: 280.44548 Molecular formula: C18H32O2

Palmitic acid IUPAC name: hexadecanoic acid Molar mass: 256.42 Molecular formula: C16H32O2

Stearic acid IUPAC name: octadecanoic acid Molar mass: 284.478 Molecular formula: C18H36O2

Oleic acid IUPAC name: (9Z)-octadec-9-enoic acid Moalr mass: 282.4614 Molecular formula: C18H34O2 Other names: (9Z)-Octadecenoic acid (Z)-Octadec-9-enoic acid cis-9-Octadecenoic acid cis-9-Octadecenoic acid

-Linolenic acid IUPAC name: all-cis-9,12,15-octadecatrienoic acid Molar mass: 278.43 Molecular formula: C18H30O2 Other names: ALA cis,cis,cis-9,12,15-Octadecatrienoic acid (9Z,12Z,15Z)-9,12,15-Octadecatrienoic acid

-Linolenic acid IUPAC name: all-cis-6,9,12-octadecatrienoic acid Molar mass: 278.43 Molecular formula: C18H30O2 Other names: GLA

Margarinic acid IUPAC name: Heptadecanoic acid Molar mass: 270.45 Molecular formula: C17H34O2 Other names: Margaric acid n-Margaric acid n-Heptadecanoic acid Heptadecylic acid n-Heptadecylic acid

Arachidic acid IUPAC name: Eicosanoic acid Molar mass: 312.5304 Molecular formula: C20H40O2 Other names: n-eicosanoic acid arachic acid

COMPARISON OF SEVERAL METHODS FOR EFFECTIVE LIPID EXTRACTION FROM MICROALGAE

Jae-Yon Lee, Chan Yoo, So-Young Jun, Chi-Yong Ahn, Hee-Mock Oh Abstract Various methods, including autoclaving, bead-beating, microwaves, sonication, and a 10% NaCl solution, were tested to identify the most effective cell disruption method. The total lipids from Botryococcus sp., Chlorella vulgaris, and Scenedesmus sp. were extracted using a mixture of chloroform and methanol (1:1). The lipid contents from the three species were 5.411.9, 7.98.1, 10.028.6, 6.18.8, and 6.810.9 g L-1 when using autoclaving, bead-beating, microwaves, sonication, and a 10% NaCl solution, respectively. Botryococcus sp. showed the highest oleic acid productivity at 5.7 mg L-1 d-1 when the cells were disrupted using the microwave oven method. Thus, among the tested methods, the microwave oven method was identified as the most simple, easy, and effective for lipid extraction from microalgae. 3.3. Fatty acid composition The major fatty acid composition of the tested microalgae was determined using a GC analysis (Table 2). In a previous report (Knothe, 2008), palmitic, stearic, oleic, and linolenic acid were recognized as the most common fatty acids contained in biodiesel. In the three tested microalgae, oleic acid (C18:1) and linoleic acid (C18:2) were commonly dominant. Oleic acid was higher in Botryococcus sp. and Scenedesmus sp. at 6.68 and 10.75 mg g -1

dw, respectively, while linoleic acid was highest in C. vulgaris at 19.79 mg g -1 dw. The daily lipid productivity of each microalgal species is shown in Table 1. The productivity of oleic acid calculated based on the daily lipid productivity was highest for Botryococcus sp. at 5.7 mg L-1 d-1. In particular, oils with a high oleic acid content have been reported to have a reasonable balance of fuel properties (Rashid et al., 2008). The properties of a biodiesel fuel, including its ignition quality, combustion heat, cold filter plugging point (CFPP), oxidative stability, viscosity, and lubricity, are determined by the structure of its component fatty esters. As such, a higher oleic acid content increases the oxidative stability for longer storage (Knothe, 2005) and decreases the CFPP for use in cold regions (Stournas et al., 1995). Therefore, among the tested microalgal species, Botryococcus sp. showed the highest oleic acid content, making it the most suitable for the production of good quality biodiesel.

WHICH ARE FATTY ACIDS OF THE GREEN ALGA CHLORELLA?

Georgi Petkov, Guillermo Garcia Abstract Fatty acid composition of three species of Chlorella were studied under conditions of photoautotrophic and heterotrophic cultivation, nitrogen starvation, and outdoor in a photobioreactor. The composition 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, a-18:3 is confirmed for Chlorella. Fatty acids with 20 carbon atoms and four or five double bonds are considered not originating from Chlorella. Other exceptions of this composition are interpreted as mixed algal culture, bacterial contamination or impurities. 3. Results and discussion The composition of fatty acids in three species of Chlorella is presented in Table 3. The comparison of quantitative composition at different cultivation conditions shows that the changes are relatively small. Here, 16:1 and 18:1 were presented with two isomers. Positional isomers of 16:1 and 18:1 were present often but not always. One of the isomers of 18:1 was oleic acid, Z-18:1. The other, namely 18:1x could be E-isomer elaidic acid or positional one (18:1). Our results show that Chlorella has quite a simple qualitative fatty acid composition compared to almost all green algae, where in addition to those found in Chlorella, 16:4 and 18:4 are identified. Furthermore, our results coincide with those obtained by several research groups, working at very different conditions, dealing with different species and strains of Chlorella. All of them report the same qualitative composition, as follows 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1, 18:2, 18:39,12,15. Trace amounts of 12:0 are present usually. There are many studies of Chlorella trying to prove that it has another fatty acid composition (see Table 2). An extremely high content of gamma-linolenic acid was found in marine Chlorella sp. by Miura et al, 1993. Up to the present moment, there is no study which confirms the presence of gamma-linolenic acid in Chlorella. Probably, the above mentioned cases concern wrongly classified genus. The classification of all marine species in Table 2 as Chlorella is doubtful. Interpreting the causes we could say that studies where 20:4 and 20:5 are rendered and no existence of 16:2 and 16:3 is found, concern some other genus (Seto et al., 1984; Yongmanitchai and Ward, 1991). The three fatty acids, 16:4, 18:3 and 18:4 appear as a result of mixed culture of green algae (Murakami et al., 1997). These acids can be found in almost all green algae. The lack of these fatty acids is rather sure chemotaxonomic marker for Chlorella.

The presence of fatty acids with odd number of carbon atoms 15:0, 17:0 and 17:1 is a firm proof that the algal culture is heavily contaminated with bacteria (DeMort et al., 1972; Podojil et al., 1978; Wright et al., 1980; Wacker et al., 2002). Cases when native Chlorella fatty acids are accompanied by 20:0, 20:1 and 20:2 can be related to impurities: not fatty acids but substances with the same retention time (Antonyan et al., 1986; Homova et al., 1986). Most often, this happens when GC analysis is performed without preliminary purification on TLC. Apart from fatty acid methyl esters, many other substances always remain in the sample, despite the precise extraction, hydrolysis and methylation as well as the direct transmethylation. Unlike the fats and oils of higher plants which are predominantly acylglycerols, the algal lipids consist of many substances so that purification of their fatty acid samples before GC is necessary.

ACID CATALYZED HOMOGENEOUS ESTERIFICATION REACTION FOR BIODIESEL PRODUCTION FROM PALM FATTY ACIDS
Donato A. G. Aranda, Rafael T. P. Santos, Neyda C. O. Tapanes, Andre Luis Dantas Ramos, Octavio Auqusto C. Antunes Abstract

This work deals with esterification of palm fatty acids to produce biodiesel in a batch reactor, using homogeneous acid catalysts, evaluating the effect of the alcohol used, presence of water, type and concentration of catalysts. Methanesulfonic and sulfuric acid were the best catalysts. Reaction with methanol showed greater yields. It was showed very clearly that the presence of water in the reaction medium showed a negative effect in the reaction velocity. Kinetic parameters were estimated and molecular modeling was performed. Protonation of the carboxylic moiety of the fatty acid were defined as rate determinant step for the reaction. Saponification reaction is an undesired reaction which may be promoted, depending on the reaction conditions and the free acid content of the vegetable oil used. The soap decreases selectivity toward biodiesel, inhibits separation of the alkyl esters and glycerol and contributes to emulsion formation during the water wash [1, 11, 14]. Esterification of FFA with low molecular weight alcoholsis another route to produce biodiesel and can be used as a pretreatment for basic transesterification reaction to convert the FFA into methyl esters and avoid saponification, especially when FFA content is higher than 1% w/w [2, 9, 10, 12]. The reaction may be represented by the following scheme:

Some authors have shown that alkyl esterification of fatty acids is faster than transesterification of triglycerides [15, 16]. This observation was assigned to the fact that alkyl esterification is a kind of one step reaction, while transesterification of triglycerides consists of three stepwise reactions, with diglycerides and monoglycerides as intermediates, and the presence of glycerol. The much higher solubility of fatty acids in low chain alcohols may also be related to this observation.

Table 1 Composition of fatty acid mixture residue 2.2 Esterification Reaction Reactions were performed in a stainless steel 600 mL batch reactor (PARR 4842), maximum pressure of 10,000 psi and equipped with a sample withdrawal, stirring and heating system. Stirring velocity was kept constant (500 rpm). Reaction mixture has consisted of 307 g of fatty acid mixture, 149 mL methanol or 215 mL ethanol, providing an alcohol/fatty acid molar ratio (A/FA) of 3. Reactants were introduced together with the appropriated catalyst mass (generally 0.1% wt/wt) and time of reaction was considered when desired temperature (generally 130C) was reached. Samples were withdrawn at 5, 10, 15, 20, 25, 30, 45 and 60 min. Reaction conversion was estimated from the FFA content of the medium by NaOH titration. 3 Results and Discussion 3.1 Esterification Reaction It can be observed that the reaction occurs in the absence of catalysts, with a conversion near 35% at 1 h. Figure 3 shows the effect of sulfuric acid concentration on the kinetic curves. A small amount of catalyst (0.01% w/w) is enough to promote the reaction. Increases in catalyst concentration accelerate the reaction progressively.

The effect of water in the reaction medium can be seen in Fig. 5. Inhibition effect can be observed mainly in ethanol reaction. Reaction inhibition by the presence of water is commonly reported in transesterification reaction, being attributed to the formation of soap, which lowers the yield of esters and renders the separation of ester and glycerol and water washing difficult, besides favoring the hydrolysis of triglycerides and FFA [1, 9, 23]. In the case of acid catalyzed esterification, soap formation is not expected, but inhibition effect was also observed [25], which may be attributed to equilibrium constraints, that is, esterification reaction has been shifted to the left, favoring the hydrolysis of the ester. Higher inhibition of ethanol reaction may be related to phase miscibility and emulsion formation.

A KINETIC ESTERIFICATION OF FREE FATTY ACIDS (FFA) IN SUNFLOWER OIL

M. Berrios, J. Siles, M.A. Martn, A. Martn

STUDY OF THE

Abstract The kinetics of the esterification of free fatty acids (FFA) in sunflower oil with methanol in the presence of sulphuric acid at concentrations of 5 and 10 wt% relative to free acids as catalyst and methanol/oleic acid mole ratios from 10:1 to 80:1 was studied. The experimental results were found to fit a first-order kinetic law for the forward reaction and a second-order one for the reverse reaction. The influence of temperature on the kinetic constants was determined by fitting the results to the Arrhenius equation. The energy of activation for the forward reaction decreased with increasing catalyst concentration from 50745 to 44 559 J/mol. Based on the experimental results, a methanol/oleic acid mole ratio of 60:1, a catalyst (sulphuric acid) concentration of 5 wt% and a temperature of 60C provided a final acid value for the oil lower than 1 mg KOH/g oil within 120 min. This is a widely endorsed limit for efficient separation of glycerin and biodiesel during production of the latter.

2. Materials and methods The sample contained the following major fatty acids: palmitic (6%), stearic (3%), oleic (17%) and linoleic (72%); all were determined by gas chromatography in accordance with UNE-EN 14331. The acid value was determined as per the UNE-55001. 3. Results and discussion By means of essays, it has been verified that the esterification reaction does not take place in catalyst absence. Tests were conducted at variable agitation speeds from 200 to 600 rpm above which this variable was found to have no further effect on the reaction rate (Fig. 2). A speed of 600 rpm was therefore subsequently used to examine the influence of the catalyst concentration, methanol/fatty acid mole ratio and temperature on the acid value. Figs. 3 and 4 show the variation of the acid value with the methanol/oleic acid mole ratio at a sulphuric acid concentration of 5% and 10%, respectively. In both cases, the reaction rate increased with increasing mole ratio. Above 60:1, however, the increase was negligible. On the other hand, an increased catalyst concentration increased the esterification rate. Thus, at a methanol/oleic acid mole ratio of 20:1, a catalyst concentration of 5% provided an acid value of 4 mg KOH/g oil within 120 min, whereas one of 10% gave 2 mg KOH/g oil in the same time.

Once a methanol/oleic acid mole ratio of 60:1 was adopted as optimal, the influence of temperature at the two catalyst concentrations studied was examined. As expected, raising the temperature increased the esterification rate (see Figs. 5 and 6). The highest rate was obtained at 60C, which is close to the boiling point of methanol at atmospheric pressure. A higher temperature obviously increased the rate further, but required using a pressure above atmospheric level or a more sophisticated experimental set-up.

High Quality Biodiesel Production from a Microalga Chlorella Protothecoides by Heterotrophic Growth in Fermenters
Han Xu, Xiaoling Miao, Qingyu Wu Abstract The aim of the study was to obtain high quality biodiesel production from a microalga Chlorella protothecoides through the technology of transesterification. The technique of metabolic controlling through heterotrophic growth of C. protothecoides was applied, and the heterotrophic C. protothecoides contained the crude lipid content of 55.2%. To increase the biomass and reduce the cost of alga, corn powder hydrolysate instead of glucose was used as organic carbon source in heterotrophic culture medium in fermenters. The result showed that cell density significantly increased under the heterotrophic condition, and the highest cell concentration reached 15.5 g L1. Large amount of microalgal oil was efficiently extracted from the heterotrophic cells by using n-hexane, and then transmuted into biodiesel by acidic transesterification. The biodiesel was characterized by a high heating value of 41 MJ kg1, a density of 0.864 kg L1, and a viscosity of 5.2104 Pa s (at 40 C). The method has great potential in the industrial production of liquid fuel from microalga. Biodiesel was obtained from heterotrophic microalgal oil by acidic transesterification (Fig. 1). The optimum process combination was 100% catalyst quantity (based on oil weight) with 56:1 molar ratio of methanol to oil at temperature of 30C, which reduced product specific gravity from an initial value of 0.912 to a final value of 0.864 in about 4 h of reaction time.

The saponification (189.3 mg KOHg1) and acid value (8.97 mg KOHg1) of the microalgal oil were determined according to the method of Vicente et al, 2004. The molecular weight of the oil was calculated from saponification and acid value as

The properties of biodiesel such as density, viscosity, flash point, cold filter plugging point, solidifying point, and heating value were measured. The elemental compositions of biodiesel were determined by a CE-440 elemental analyzer (Peng et al., 2001). The composition of the biodiesel was derivatized and analyzed by gas chromatographicmass spectrometric analysis. Gas chromatography was performed on a 0.25mm (i.d.)30m fused silica column lined with a 0.25 _m film of polyethylene glycol (VF-5ms, from VARIAN, America). Samples (0.2_L) were injected in split mode (split/column flow ratio 30:1). The column head pressure of the carrier gas (helium) was 3 kPa at the initial oven temperature, and its flow rate 1.0mLmin1. The injection temperature was 290 C; the oven temperature was 100 C for 2 min, rose to 300 C over 20 min and was held at this temperature for 20 min (total run time 42 min). The GCMSapparatus was linked to a PC running software for data acquisition and processing.

3.5. Biodiesel produced from heterotrophic Chlorella To assess the potential of biodiesel as a substitute of diesel fuel, the properties of biodiesel such as density, viscosity, flash point, cold filter plugging point, solidifying point, and heating value were determined. A comparison of these properties of diesel fuel (Ma and Hanna, 1999; Lang et al, 2001; Al-Widyan and Al-Shyoukh, 2002; Antolin et al, 2002; Vicente et al, 2004), biodiesel from microalgal oil and ASTM biodiesel standard is shown in Table 4. Most of these parameters comply with the limits established by ASTM related to biodiesel quality (Antolin et al, 2002). The physical and fuel properties of bidiesel from microalgal oil in general were comparable to those of diesel fuel. The biodiesel from microalgal oil showed much lower cold filter plugging point of 11 C in comparison with the diesel fuel (Table 4). The gas chromatograph of biodiesel is shown in Fig. 5. The fatty acid methyl esters (FAMEs) of the biodiesel are presented in Table 5. There were nine FAMEs derivatized in the biodiesel, and the most abundant composition was oleic acid methyl ester with the content of 60.84%. Oleic acid methyl ester, octadecadienoic acid methyl ester, and octadecanoic acid methyl ester are 18 carbon acid methyl esters, and the total content of these three FAMEs was over 80%. This resulted in the high quality of the biodiesel.

A Review on Biodiesel Production, Combustion, Emissions and Performance

Abstract This article is a literature review on biodiesel production, combustion, performance and emissions. This study is based on the reports of about 130 scientists who published their results between 1980 and 2008. As the fossil fuels are depleting day by day, there is a need to find out an alternative fuel to fulfill the energy demand of the world. Biodiesel is one of the best available sources to fulfill the energy demand of the world. More than 350 oil-bearing crops identified, among which some only considered as potential alternative fuels for diesel engines. The scientists and researchers conducted tests by using different oils and their blends with diesel. A vast majority of the scientists reported that short-term engine tests using vegetable oils as fuels were very promising but the long-term test results showed higher carbon built up and lubricating oil contamination resulting in engine failure. They concluded that vegetable oils, either chemically altered or blended with diesel to prevent the engine failure. It was reported that the combustion characteristics of biodiesel are similar as diesel and blends were found shorter ignition delay, higher ignition temperature, higher ignition pressure and peak heat release. The engine power output was found to be equivalent to that of diesel fuel. In addition, it observed that the base catalysts are more effective than acid catalysts and enzymes. Production of biodiesel Researchers and scientists had developed different methods for biodiesel production from different bio fuels. A brief review of these methods has presented here. Most of the researchers/scientists reported that the production of biodiesel was more when the process was used a catalyst. Ahn et al. [1] followed a two-step reaction process to produce biodiesel. Using this method canola methyl ester (CME), rapeseed methyl ester (RME), linseed methyl ester (LME), beef tallow ester (BTE) and sunflower methyl ester (SME), synthesized in a batch reactor using sodium hydroxide, potassium hydroxide and sodium

methoxide as catalysts. Cvengro and Povaz [2] described biodiesel production by using two-stage lowtemperature transesterification of cold pressed rapeseed oil with methanol at temperatures up to 70C. A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed by Masaru et al. [3]. In the same year, Uosukainen et al. [4] presented statistical and experimental design to evaluate interdependence of process variables in enzymatic transesterification. The authors also studied the alcoholysis of rapeseed oil methyl ester (biodiesel). Fangrui and Hanna [5] had reviewed the biodiesel production. Samukawa et al. [6] investigated the effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production from soybean. Ikwuagwu et al. [7] discussed the production of biodiesel using rubber seed oil. The effect of three principal variables namely molar ratio of methanol to oil, amount of catalyst and reaction temperature on the yield of acid-catalyzed production of methyl ester (biodiesel) from crude palm oil had been studied by Crabbe et al. [8]. Transesterification reaction of rapeseed oil in supercritical methanol was investigated without using any catalyst by Saka and Kusdiana [9]. Yuji Shimada et al. [10] studied the enzymatic alcoholysis for biodiesel fuel production. Pizarro and Park [11] studied the production of biodiesel fuel from vegetable oils contained in waste activated bleaching earth. Shieh et al. [12] optimized the biodiesel production from soybean by using response surface technology. Zhang et al. [13] reported that the acid-catalyzed process using waste cooking oil has proved to be technically feasible with less complexity than the alkali-catalyzed process using waste cooking oil. Fatty acid methyl ester (FAME) production from waste activated bleaching earth discarded by the crude oil refining industry was investigated by Kojima et al. [14] using fossil fuel as a solvent in the esterification of triglycerides. Kusdiana and Saka [15] discussed the effects of water on biodiesel fuel production by supercritical methanol treatment. Tashtoush et al. [16] conducted an experimental study on evaluation and optimization of conversion of waste animal fat into biodiesel. Ghadge and Raheman [17] studied biodiesel production from mahua (Madhuca indica) oil having high free fatty acids. Van Gerpen [18] discussed the effects of reaction time, reaction temperature on quality and quantity of esters. It is concluded that a trade-off between reaction time and temperature as reaction completeness is the most critical fuel quality parameter [18]. Xu et al. [19] proposed a simplified model to describe the reaction kinetics of the biodiesel production. Cao et al. [20] had carried out transesterification of soybean oil in supercritical methanol in the absence of catalyst. Ramadhas et al. [21] studied biodiesel production from high free fatty acid rubber seed oil. They developed a two-step transesterification process to convert the high free fatty acid oils to its mono-esters. The major factors affect the conversion efficiency of the process such as molar ratio, amount of catalyst, reaction temperature and reaction duration analyzed [21]. Karmee and Chadha [22] prepared biodiesel from the Pongamia pinnata by transesterification in the presence of potassium hydroxide as catalyst. Ghadge and Raheman [23] discussed the preparation of biodiesel from high free fatty acid oils by using response surface methodology. In the same year, Canoira et al. [24] presented a process to convert the Jojoba oil wax to biodiesel by Transesterification with methanol. Waste frying oils transesterification was studied by Felizardo et al. [25] with the purpose of achieving the best conditions for biodiesel production. In the same year, Miao and Wu[26] introduced an integrated method for the production of biodiesel from microalgal oil. Zhu et al. [27] produced biodiesel from jatropha curcas oil using a heterogeneous solid super base catalyst (calcium oxide). Meher et al. [28] reviewed the technical aspects of biodiesel production by transesterification. Al-Zuhair et al. [29] discussed the effect of fatty acid concentration and water content on the production of biodiesel. Xue et al. [30] had developed a new method for preparing raw material for biodiesel. Production of fatty acid methyl esters from crude tobacco seed oil (TSO) having high free fatty acids (FFA) was investigated by Veljkovic et al. [31]. Royon et al. [32] studied the enzymatic production of biodiesel by methanolysis of cottonseed oil. A two-phase membrane reactor had developed to produce biodiesel from canola oil by Dube et al. [33]. Li et al. [34] optimized the whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology. Transesterification reaction of used frying oil by means of ethanol, using sodium hydroxide, potassium hydroxide, sodium methoxide, and potassium methoxide as catalysts, was studied by Encinar et al. [35]. Issariyakul et al. [36] studied the production of biodiesel from waste fryer grease using mixed methanol/ethanol system. Chisti [37] discussed the biodiesel production from microalgae. A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst has developed for biodiesel fuel production by plant oil methanolysis by Hama et al. [38]. Fatty acids

methyl esters had prepared by Hernando et al. [39] under microwave irradiation, using homogeneous catalysis, either in batch or in a flow system. They reported that the process using microwaves irradiation proved to be a faster method for alcoholysis of triglycerides with methanol, leading to high yields of fatty acid methyl ester [39]. Response surface methodology (RSM) based on central composite rotatable design (CCRD) was used to optimize the three important reaction variables namely methanol quantity, acid concentration and reaction time for reduction of free fatty acid content of the oil to around 1% by Tiwari et al. [42].

MODELING AND SIMULATION OF REACTION KINETICS FOR BIODIESEL PRODUCTION

Lawrence Turner, Timothy Transesterification reactions can be base-catalyzed, acid catalyzed, or enzymatic. The base-catalyzed reaction takes about one hour at room temperature. It suffers from competing saponification reactions, which convert the same ingredients as well as any free fatty acids to soap. Acid-catalyzed and enzymatic transesterification require three to four days to complete. The acid-catalyzed reaction also requires heat. There is no competing saponification reaction with the acid-catalyzed and enzymatic reactions. In fact, even free fatty acids are converted to biodiesel by esterification. The associated acid-catalyzed esterification reaction requires only about two hours completion. A combined strategy called the two-stage process can be used maximize the amount of biodiesel produced, while minimizing the amount of produced. The first stage is acid-catalyzed esterification of the free fatty acids. This followed by base-catalyzed transesterification. This approach is especially effective for waste vegetable oil and animal fats, which have high free fatty acid content.

PROYECTO DE NORMA TCNICA COLOMBIANA NTC 1438 (Sexta actualizacin)

1. OBJETO Esta norma establece las especificaciones que deben cumplir y los mtodos de ensayos que se deben usar para determinar los parmetros definidos para los combustibles utilizados en motores tipo diesel disponibles en Colombia (vase el numeral 3). 3. DEFINICIONES 3.1 Biodiesel. Son mezclas de mono - alquil steres de los cidos grasos de cadena larga derivados de aceites vegetales y grasas animales. 3.2 Diesel corriente. Es una mezcla de hidrocarburos entre 10 tomos y 28 tomos de carbono formada por fracciones combustibles proveniente de diferentes procesos de refinacin del petrleo tales como destilacin atmosfrica y ruptura cataltica, que se utiliza como combustible en motores tipo diesel y que puede contener Biodiesel. 3.3 Diesel extra. Mezcla de hidrocarburos similar al diesel corriente pero con un menor contenido de azufre, menor viscosidad, menor punto final de ebullicin y que puede contener Biodiesel. 3.4 Mezclas biodiesel - diesel, BXX. Son mezclas de biodiesel con diesel en diferentes proporciones. La letra B representa el biodiesel y XX representa su porcentaje en volumen (% vol.) en la mezcla final. 4. REQUISITOS 4.1 Los combustibles Diesel extra " y Diesel corriente deben cumplir los requisitos indicados en la Tabla 1. 4.2 El biodiesel B100 debe cumplir con los requisitos indicados en la Tabla 1 del DE 100-04 (ICONTEC) Biodiesel para motores diesel. 5. TOMA DE MUESTRAS Y CRITERIOS DE ACEPTACIN O RECHAZO 5.1 TOMA DE MUESTRAS La toma de muestras se efecta de acuerdo con lo indicado en la NTC 1647, tomando mnimo un volumen equivalente a 20 L (20 dm3).

Palmitoleic acid IUPAC name: hexadec-9-enoic acid Molar mass: 254.408 Molecular formula: C16H30O2 Other names: Palmitoleic acid cis-Palmitoleic acid 9-cis-Hexadecenoic acid

7,10-Hexadecadienoic acid IUPAC name: hexadeca-7,10-dienoic acid Molar mass: Molecular mass: C16H28O2 7,10,13-hexadecatrienoic acid IUPAC name: hexadeca-7,10,13-trienoic acid Molar mass: 250.38 Molecular formula: C16H26O2

BIODIESEL PROCESSING AND PRODUCTION

Jon Van Gerpen Abstract Biodiesel is an alternative diesel fuel that is produced from vegetable oils and animal fats. It consists of the monoalkyl esters formed by a catalyzed reaction of the triglycerides in the oil or fat with a simple monohydric alcohol. The reaction conditions generally involve a trade-off between reaction time and temperature as reaction completeness is the most critical fuel quality parameter. Much of the process complexity originates from contaminants in the feedstock, such as water and free fatty acids, or impurities in the final product, such as methanol, free glycerol, and soap. Processes have been developed to produce biodiesel from high free fatty acid feedstocks, such as recycled restaurant grease, animal fats, and soapstock.

Freedman compared both crude and refined vegetable oils as feedstocks and found that the yield of methyl esters was reduced from 93% to 98% for the refined oil to 67% to 86% for the crude oil. This was attributed mostly to the presence of up to 6.66% free fatty acids in the crude oil, although phospholipids were also suggested as a source of catalyst destruction. In a study of the effects of phospholipids on biodiesel production, Van Gerpen and Dvorak [20] found that phosphorus compounds in the oil did not carry over into the methyl esters, and while yield was reduced by 35% for phosphorus levels above 50 ppm, this was due mostly to added difficulty separating the glycerol from the esters. Fig. 2 shows a schematic diagram of the processes involved in biodiesel production. Alcohol, catalyst, and oil are combined in a reactor and agitated for approximately 1 h at 60C. Smaller plants often use batch reactors [27], but most larger plants (>4 million liters/year) use continuous flow processes involving continuous stirred-tank reactors (CSTR) or plug flow reactors [28]. The reaction is sometimes done in two steps (as suggested by the patent literature discussed earlier). In this system, approximately 80% of the alcohol and catalyst is added to the oil in a first stage CSTR. Then the reacted stream from this reactor goes through a glycerol removal step before entering a second CSTR. The remaining 20% of the alcohol and catalyst are added in this reactor. This system provides a very complete reaction with the potential of using less alcohol than single-step systems.

Following the reaction, the glycerol is removed from the methyl esters. Due to the low solubility of glycerol in the esters, this separation generally occurs quickly and may be accomplished with either a settling tank or a centrifuge. The excess methanol tends to act as a solubilizer and can slow the separation. However, this excess methanol is usually not removed from the reaction stream until after the glycerol and methyl esters are separated due to concern about reversing the transesterification reaction. Water may be added to the reaction mixture after the transesterification is complete to improve the separation of glycerol [27,29]. Saka and Kusiana [3034] claim that it is possible to react the oil and methanol without a catalyst, which eliminates the need for the water washing step. However, temperatures of 300C 350C and methanol to oil molar ratios of 42:1 are required. While the reaction only requires 120 s, the purity of the final product needs to be fully characterized, and formation of nonmethyl ester compounds in significant amounts is possible. Dasari et al. [33] measured reaction rates without catalysts at temperatures of 120C to 180C. They noted the difficulty of reproducing reaction kinetics results of other researchers [31,35] and attributed it to catalytic effects at the surfaces of the reaction vessels and noted these effects would be exacerbated at higher temperatures. Boocock et al. [3739] have developed a novel technique for accelerating the transesterification reaction rate. During its early stages, the transesterification reaction is limited by the low solubility of the alcohol, especially methanol, in the oil. Boocock proposed the addition of a cosolvent to create a single phase, and this greatly accelerates the reaction so that it reaches substantial completion in a few minutes. The technique is applicable for use with other alcohols and for acid-catalyzed pretreatment of high free fatty acid feed stocks. The primary concerns with this method are the additional complexity of recovering and recycling the cosolvent although this can be simplified by choosing a cosolvent with a boiling point near that of the alcohol

being used. Additional concerns have been raised about the hazard level associated with the cosolvents most commonly proposed, tetrahydrofuran and methyl tertiary butyl ether. Returning to Fig. 2, after separation from the glycerol, the methyl esters enter a neutralization step and then pass through a methanol stripper, usually a vacuum flash process or a falling film evaporator, before water washing. Acid is added to the biodiesel to neutralize any residual catalyst and to split any soap that may have formed during the reaction. Soaps will react with the acid to form water soluble salts and free fatty acids. The salts will be removed during the water washing step, and the free fatty acids will stay in the biodiesel. The water washing step is intended to remove any remaining catalyst, soap, salts, methanol, or free glycerol from the biodiesel. Neutralization before washing reduces the water required and minimizes the potential for emulsions to form when the wash water is added to the biodiesel. Following the wash process, any remaining water is removed from the biodiesel by a vacuum flash process. The glycerol stream leaving the separator is only about 50% glycerol. It contains some of the excess methanol and most of the catalyst and soap. In this form, the glycerol has little value and disposal may be difficult. The methanol content requires the glycerol to be treated as hazardous waste. The first step in refining the glycerol is usually to add acid to split the soaps into free fatty acids and salts. The free fatty acids are not soluble in the glycerol and will rise to the top where they can be removed and recycled. The salts remain with the glycerol although depending on the chemical compounds present, some may precipitate out. Mittelbach describes a process for esterifying these free fatty acids and then returning them to the transesterification reaction stream [40]. One frequently touted option is to use potassium hydroxide as the reaction catalyst and phosphoric acid for neutralization so that the salt formed is potassium phosphate, which can be used for fertilizer. After acidulation and separation of the free fatty acids, the methanol in the glycerol is removed by a vacuum flash process or another type of evaporator. At this point, the glycerol should have a purity of approximately 85% and is typically sold to a glycerol refiner. The glycerol refining process takes the purity up to 99.5% to 99.7% using vacuum distillation or ion exchange processes. The methanol that is removed from the methyl ester and glycerol streams will tend to collect any water that may have entered the process. This water should be removed in a distillation column before the methanol is returned to the process. This step is more difficult if an alcohol such as ethanol or isopropanol is used that forms an azeotrope with water. Then a molecular sieve is used to remove the water. Special processes are required if the oil or fat contains significant amounts of free fatty acids (FFAs). Used cooking oils typically contain 27% FFAs, and animal fats contain from 5% to 30% FFAs. Some very low quality feedstocks, such as trap grease, can approach 100% FFAs. When an alkali catalyst is added to these feedstocks, the free fatty acids react with the catalyst to form soap and water, as shown in the reaction below:

Up to about 5% FFAs, the reaction can still be catalyzed with an alkali catalyst, but additional catalyst must be added to compensate for the catalyst lost to soap. The soap that is created during the reaction is either removed with the glycerol or is washed out during the water wash. When the FFA level is above 5%, the soap inhibits separation of the methyl esters and glycerol and contributes to emulsion formation during the water wash. For these cases, an acid catalyst, such as sulfuric acid, can be used to esterify the free fatty acids to methyl esters, as shown in the following reaction:

This process can be used as a pretreatment to convert the FFAs in high FFA feedstocks to methyl esters and thereby reduce the FFA level. Then the low FFA pretreated oil can be transesterified with an alkali catalyst to convert the triglycerides to methyl esters. Keim [9] describes using this approach to convert palm oil containing 50.8% free fatty acids to methyl esters. Methanol (77% of the weight of oil) and sulfuric acid (0.75% of the weight of oil) were added to the oil while stirring at 69C for 1 h. After neutralization, 1.25% sodium methoxide was added,

and the mixture was stirred for an additional hour at 50C. Analysis showed a yield of 97% but a residual acid value equivalent to about 5% palmitic acid. The incomplete reaction was probably due to water in the reactant mixture. As shown in the reaction, water is formed, and if it accumulates, it can stop the reaction well before completion. Kawahara and Ono [42] propose allowing the alcohol to separate from the pretreated oil or fat following the reaction. Removal of this alcohol also removes the water formed by the esterification reaction and allows for a second step of esterification or proceeding directly to alkali-catalyzed transesterification. Jeromin et al. [41] has described using acidic ion exchange resins in a packed bed for the pretreatment. References [9] G.I. Keim, Treating fats and fatty oils, U.S. Patent No. 2,383,601 August. 28, 1945 [20] J.H. Van Gerpen, B. Dvorak, The effect of phosphorus level on the total glycerol and reaction yield of biodiesel, Bioenergy 2002, The 10th Biennial Bioenergy Conference, Boise, ID, September 2226, 2002 [27] W.D. Stidham, D.W. Seaman, M.F. Danzer, Method for preparing a lower alkyl ester product from vegetable oil, US Patent No. 6,127,560 October 3, 2000 [29] T. Wimmer, Process for the production of fatty acid esters of lower alcohols, US Patent No. 5,399,731 21 March 21,1995 [42] Y. Kawahara, T. Ono, Process for producing lower alcohol esters of fatty acids, US Patent No. 4,164,506 August 14, 1979

US PATENT 4164506 - PROCESS FOR PRODUCING LOWER ALCOHOL ESTERS OF FATTY ACIDS
SUMMARY OF THE INVENTION The inventions of the present invention have therefore made intensive research to achieve a process for obtaining a high quality lower alcohol ester of a fatty acid and glycerol from an unrefined fat, with a high yield, by solving the above-mentioned defects of the conventional methods, and thus completed the present invention. In accordance with the present invention, there is provided a process for producing lower alcohol esters of fatty acids which comprises steps including: (1) esterifying free fatty acids of unrefined fats with a lower alcohol in an amount larger than the amount soluble in the fats, in the presence of an acid catalyst, or esterifying free fatty acids of unrefined fats with a lower alcohol in the

presence of an acid catalyst and then adding the lower alcohol in an amount larger than the amount soluble in the fats; (2) separating the product mixture into the fat layer and the lower alcohol layer, so that the latter layer may be removed; and (3) effecting the interesterification reaction between the resulting refined fats and a lower alcohol with an alkali catalyst. After the esterification of the free fatty acid using an acid catalyst and the separation of the alcohol layer in the above-mentioned process, the procedure comprising adding again the lower alcohol in an amount larger than its soluble amount in the fat, stirring, settling, separating and removing the alcohol layer, may be further repeated at least once, and the resulting refined fats and the lower alcohol are subjected to the interesterification reaction using an alkali catalyst to thereby give a lower alcohol ester of the fatty acid having a further improved quality. The fats and oils used as raw materials in the present invention are not refined or not completely refined. Accordingly, they contain free fatty acids, polypeptides, phospholipids, and other impurities. The solubility of an alcohol in the fat depends on various conditions. In the case of methanol, the solubility is from 12 to 15% by weight in the fat at 50 C. Accordingly, in the present invention, methanol is added in an amount larger than this solubility. More specifically, it is preferred to add methanol in an amount of 20 to 30% by weight based on the weight of the fat. The esterification reaction may be carried out in a sealed system after adding a lower alcohol in excess. However, in order to remove water generated during the reaction, it is more preferred that methanol is charged in excess at the start of the reaction and then further methanol is blown into the reaction system at the same rate as that at which methanol is being distilled out. The esterification of free fatty acids by the use of an acid catalyst is carried out at a temperature in the range of from 60 to 120 C. In order to restrain the interesterification reaction of the fat component, however, a low temperature ranging from 65 to 70 C. is more preferred. The most characteristic feature of the process for producing lower alcohol esters of fatty acids in accordance with the present invention resides in the following points. It is different from the conventional solvent extraction method which merely removes free fatty acids by means of the solubility difference, and the esterification method in which free fatty acids are esterified with a lower alcohol into fatty acid esters so as to reduce the acid value. According to the process of the present invention, not only free fatty acids in unrefined fats are esterified to reduce their acid value, but also simultaneously the fats are treated with an acid catalyst for the purpose of the esterification and impurities other than the free fatty acids, such as polypeptides and phospholipids, are dissolved in the lower alcohol layer which is present in excess or is later added, and thus removed from the fats. The removal of these impurities is difficult without the acid treatment of the fats. In other words, in the conventional solvent extraction method, these impurities have poor solubility in a solvent and they are hardly removed from the fat as they are. In the process of the present invention, on the other hand, the impurities such as polypeptides and phospholipids are decomposed while the fatty acid is being esterified under the acidic condition of sulfuric acid and their solubility in a lower alcohol is increased. As the result, the effect of removing impurities is remarkably enhanced in the solvent extraction step. Moreover, as the unreacted free fatty acid is distributed also to the lower alcohol layer to a certain extent, so it is also advantageous for the removal of the impurities. After the removal of the alcohol layer separated at the end of the esterification, the lower alcohol is added to the fat in an amount larger than its soluble amount in the fat (preferably 10 to 30% by weight based on the weight the fat), stirred, settled, and subjected to a layer separation procedure so as to remove the excess lower alcohol. When the cycle of these steps is repeated, the fats are improved in their quality. It is possible to re-use the resulting alcohol layer of this procedure in the step of adding a lower alcohol to the starting fats. In accordance with the present invention, it is now possible to obtain a fatty acid ester and glycerol of extremely high quality from unrefined fats and oils containing various impurities by employing the process comprising esterification, layer separation, washing with lower alcohol, layer separation and interesterification. As to the byproduced glycerol, the recovery yield of glycerol is improved because the loss caused by incorporation of the fat and the resulting emulsion is minimized.

Example A four-necked flask of a 2 l-capacity equipped with a stirrer, a thermometer, a methanol blowing port and a condenser for distillates is charged with 1,000 g of a raw coconut oil having an acid value of 7.7, 205 g of methanol and 1 g of 98% concentrated sulfuric acid. The mixture is heated, and after methanol starts reflux, further methanol is blown into the flask at a constant rate so as to keep the methanol in the flask in the same amount as the initially charged water and methanol. The esterification reaction is carried out for 3 hours at a temperature of 65 to 67 C. At 3 hours after the blowing of methanol started, the blowing is stopped and the reaction system is then cooled to 50 C. Thereafter, the stirring is also stopped and the reaction system is allowed to stand, whereupon the upper methanol layer and the lower fat layer separate in a volume ratio of about 1:10. After the methanol layer is removed, 205 g of methanol is further added to the fat layer, and the mixture is stirred at 50 C. to wash the fat layer with it. In the same way as above, the methanol layer is separated. The acid value is lowered down to 0.3 in this instance. 1.74 g of caustic soda as an alkali catalyst and 75.6 g of methanol are added to the resulting fat and subjected to the interesterification reaction at about 50 C. for about 1 hour. The reaction mixture is allowed to stand, and the lower glycerol layer is removed. Then 1.74 g of caustic soda and 15.6 g of methanol are added and subjected to the interesterification for about 30 minutes. The reaction mixture is again allowed to stand to separate and remove the glycerol layer. There is thus obtained a coconut fatty acid methyl ester. For comparison, the esterification of the fatty acid is carried out in substantially the same manner as above, except that the blowing of methanol, subsequent separation and removal of the methanol layer and washing of the fat with methanol are not effected. Then, the interesterification reaction is similarly performed between the resulting fat and methanol in the presence of the alkali catalyst to give a coconut fatty acid methyl ester and crude glycerol (pre-esterification method).

US PATENT 5399731 - PROCESS FOR THE PRODUCTION OF FATTY ACID ESTERS OF LOWER ALCOHOLS
SUMMARY OF THE INVENTION It was surprisingly found that the aforementioned disadvantages can be avoided and that the requirement mentioned above can be met by carrying out the transesterification of fatty acid glycerides with 1.10 to 1.80 mols of a lower alcohol based on 1 mol of fatty acid bound as glyceride in the presence of an alkali or alkaline earth metal compound. Preferably, the transesterification is carried out in the presence of sodium hydroxide or potassium hydroxide. Subsequent to the completed transesterification and the separation of the glycerol phase, 0.1 to 5 percent based on the ester phase of water or a diluted organic or inorganic acid are added with stirring to remove residual impurities such as in particular glycerol or catalyst residues, the amount depending on the amount of alkali or alkaline earth metal remaining in the ester phase and preferably ranging between 0.3 and 3 percent, and withdrawing the heavy phase after it has settled. The transesterification can be carried out in one or several stages. The fatty acid glyceride is either transesterified with the total amount of lower alcohol and catalyst or a first stage is carried out with only a portion of the amount of lower alcohol and catalyst required for the transesterification. The remaining amount(s) of lower alcohol and catalyst required for the transesterification are added in the same manner, subsequently to completed settling and separation of a glycerol phase, in a second stage or in several further stages. The two- and multiple-stage operations entail the advantage of a further reduction of the alcohol excess. If the transesterification is carried out in a two-stage operation, 6/10 to 9/10 of the total amount of lower alcohol and catalyst required are preferably added in the first stage, while 1/10 to 4/10 are added in the second stage. In two- or multiple-stage operation, water can be added immediately after the second or the respective last stage, i.e., without previous separation of the glycerol phase formed in the second or last stage. DESCRIPTION OF THE PREFERRED EMBODIMENTS Transesterification according to the process of the present invention is preferably carried out at ambient temperatures of about +5 C. to +40 C. and atmospheric pressure and can be carried out in any given open or closed container of any given dimensions preferably provided with a discharge orifice in the bottom. At batch volumes of about 2,000 liters, the required stirring manipulations can be carried out manually or automated if required.

There are two substantial characterizing features of the process according to the invention. Firstly, the amount of catalyst used, whereby any given high degrees of transesterification and a trouble-free sedimentation and separation of the glycerol phase can be achieved at ambient temperature and atmospheric pressure and low alcohol excess at high degree of contamination and high content in free fatty acids in the fatty acid glycerides used. Secondly, the addition of water or an organic or inorganic acid after transesterification, whereby a troublefree elimination of the catalyst residues from the ester phase and other residual impurities such as glycerol, phosphatides and the like from the ester phase is made possible. Known processes in which the removal of the catalyst residues from the ester phase is effected by one or several washing(s) with water or acids, result in great difficulties due to the formation of emulsions of fatty acids in the free state and subsequent elaborate phase separation and drying. In contrast, the process according to the invention provides for the sedimentation of the heavy phase containing the added water and the aforementioned impurities and the catalyst residues without the formation of emulsions and the ester phase can be drawn off after several hours. The addition of water characteristic for the present invention is not comparable to the conventional washing operations of the known processes, which is already evident from the small amounts of water preferably used according to the invention, i.e., of 0.3 to 3 percent based on the ester phase. Instead, what is involved in the present case is the hydration of the anhydrous catalyst and glycerol residues present in the fatty acid esters with the added water. In contrast to the washing operations according to the known processes, the fatty acid esters are virtually anhydrous after the addition of water according to the invention and the settling of the heavy phase. If required, the separation of the heavy phase can be accelerated by means of a coalescence separator. Instead of water, a diluted organic or inorganic acid such as acetic acid, oxalic acid, hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid could be used, namely, in amounts of from 0.1 to 5 percent, preferably of from 0.3 to 3 percent. The concentration of the acid is selected so that it is at least equivalent, and exceeds by up to 100 percent, the amount of transesterification catalyst remaining in the ester phase. This embodiment is mainly used if very small amounts of residual catalyst are contained in the ester phase. The glycerol phase accumulating in the process according to the invention may contain large amounts of soaps particularly when using fatty acid glycerides with high proportions of fatty acids in the free state. The glycerol phase as well as the heavy phase accumulating after the addition of water which also contains glycerol and soaps, can conveniently be processed with concentrated phosphoric acid, such as it is described in the "Process for the Treatment of the Glycerol Phase Accumulating in the Transesterification of Fats and Oils with Low Alcohols" (Austrian patent application A 2357/89) of the applicant. The process according to the invention does not preclude that the fatty acid esters thus produced, should the intended use require it, be subjected to a further purification by conventional measures. For instance, measures such as vacuum distillation, elimination of residual amounts of lower alcohols by means of evaporation or blowing out, additional drying by means of silica gel, molecular sieves, coalescence aids or the like, removal of higher melting portions of fatty acid esters by freezing, color and odor improvement or reduction of the peroxide number by treatment with bleaching earths and the like or the addition of additives such as agents for lowering the solidification point, for improving viscosity, for inhibiting corrosion, for protection against oxidation, improving the cetane number and the like, may be used so as to adapt the produced fatty acid esters to the respective intended use in a known manner. The advantages of the process according to the invention particularly reside in the following facts. The operation can be carried out at low excesses of lower alcohols at ambient temperatures of +5 to +40 C. and atmospheric pressure. The oils and fats used need not be subjected to any purification and can contain up to 20 percent and more of fatty acids in the free state in addition to slimy substances, phosphatides and other impurities. Any given high degree of transesterification, rendering any further purification, for instance, by means of distillation, redundant for most applications, can be obtained depending on the preferably cited amounts of lower alcohol and transesterification catalyst. For instance, if the fatty acid esters are to be used as diesel fuels, no washing operations with water or acids and no ion exchangers are required for removing the catalyst residues. The transesterification can be carried out, if necessary, at temperatures of +5 C. and less, which is particularly convenient with fatty acid glycerides with sensitive highly unsaturated fatty acids such as alpha and gamma linoleic acid, eicosapenta and docosahexaenoic acids and the like because it prevents isomerizations, which is significant for the use of such fatty acid esters for pharmaceutical, dietetic and cosmetic purposes. Also, the fatty acid esters can be produced in a technically extremely simple manner.

EXAMPLE 1 A solution of 1.83 g (0.033 mols) of potassium hydroxide in 19 ml of methanol is added to 100 g of deslimed and deacidified (acid number 0.07) rapeseed oil and stirred in a beaker of 250 ml cubical content by means of a magnetic agitator for 15 minutes. After being left to stand for one hour, the glycerol phase accumulated on the bottom of the beaker is drawn off, e.g., by sucking up in a pipette. 0.5 l of water are added and stirring is continued for 10 minutes. After having been left standing for 12 hours, the supernatant is decanted. It consists of virtually pure rapeseed fatty acid methyl ester without any detectable fatty acid mono-, di- or triglycerides and an ash content of 0.004 percent. EXAMPLE 2 In a container with a cubical capacity of 2000 liters equipped with an agitator and a discharge device in the bottom, 1618 kg of unrefined rapeseed oil with a content of 2.25 percent of fatty acids in the free state are charged. A solution of 27.8 kg of industrial grade potassium hydroxide (corresponding to 24.5 kg of 100 percent KOH) in 240 l of methanol is added and stirring is continued for 20 minutes. After a sedimentation period of three and one-half hours, the glycerol phase on the bottom of the container is drained off and stirring is resumed after the addition of a solution of 6.9 kg of industrial grade potassium hydroxide (corresponding to 6.1 kg of 100 percent KOH) in 60 l of methanol and continued for 20 minutes again. After that, 80 kg of water are immediately added and stirring is continued for another five minutes. After being left standing overnight, the heavy phase is drained off from the bottom of the container. The supernatant is suitable for use as a diesel fuel without any further treatment. It contains less than 1.5 percent residual fatty acid glycerides and 0.008 percent ash. Ricinoleic acid IUPAC name: (9Z,12R)-12-Hydroxyoctadec-9-enoic acid Molar mass: 298.461 Molecular formula: C18H34O3 Other names: R12-Hydroxy-9-cis-octadecenoic acid

BIODIESEL PRODUCTION FROM WASTE COOKING OIL: 1. PROCESS DESIGN AND TECHNOLOGICAL ASSESSMENT
Y. Zhang a, M.A. Dub_e a, D.D. McLean, M. Kates 4.1. Alkali-catalyzed process using virgin vegetable oil (process I) 4.1.1. Transesterification A continuous alkali-catalyzed process flowsheet using virgin oil was developed (Fig. 2). The reaction was carried out with a 6:1 molar ratio of methanol to oil, 1% sodium hydroxide (based on oil), 60 _C and 400

kPa. Fresh methanol (stream 101 at 117 kg/h), recycled methanol (stream 1201 at 111 kg/h) and anhydrous sodium hydroxide (stream 103 at 10 kg/h) were mixed prior to being pumped into reactor R101 by pump P-101. Virgin vegetable oil (stream 105) was heated in exchanger E-101 before entering R101. In R-101, 95% of oil was assumed to be converted to FAME, producing glycerol as a by-product. Stream 106 from the reactor was introduced to methanol distillation T-201. 4.1.2. Methanol recovery In T-201, five theoretical stages and a reflux ratio of 2 were used to obtain a good separation between methanol and other components. Stream 201 was a pure methanol distillate, containing 94% of the total methanol in stream 106. Vacuum distillation was used to keep the bottom temperature under 150 _C. Pure methanol (stream 1201) was mixed with fresh make-up methanol (stream 101B) and then charged back into reactor R-101. Bottom stream 202 was sent to washing column T-301 after being cooled in exchanger E-201 to 60 _C. 4.1.3. Water washing The purpose of this step was to separate the FAME from the glycerol, methanol and catalyst. Although separation using a gravity settler was proposed by Krawczyk (1996), a complete separation could not be achieved based on our simulation results. Consequently, a water washing column (T-301) with four theoretical stages was used in this study (Connemann and Fischer, 1998). The FAME in stream 203 was separated from the glycerol, methanol and catalyst by adding 11 kg/h water (25 _C). The amounts of unconverted oil, methanol and water in stream 301A were all less than 6%. All of the glycerol remained in the bottom stream 303 (128 kg/h), which contained 81% glycerol, 8% water, 3% methanol and 9% sodium hydroxide. 4.1.4. FAME purification In order to obtain a final biodiesel product adhering to ASTM specifications (greater than 99.6% pure), FAME distillation T-401 with four theoretical stages and a reflux ratio of 2 were used. Stream 301A from T-301 was forwarded to T-401. T-401 was operated under vacuum to keep temperatures low enough to prevent degradation of the FAME. A partial condenser was used to provide easy separation of the FAME from water and methanol in the column overhead. Water and methanol were removed as vent gases (stream 401A). FAME product (99.65% purity) was obtained in stream 401 as a liquid distillate (194 _C and 10 kPa). Unconverted oil remained at the bottom of T-401. Since only a small amount of unconverted oil (52 kg/h) was left, it was treated as a waste. When oil conversion in reactor R-101 was low and oil recycling was necessary for waste reduction, a cooler and a pump were required to pump the unconverted oil back to the transesterification reactor. Superheated high pressure steam was the heating medium for the reboiler. When the UNIQUAC model rather than NRTL was employed, the load in T-401 increased. Accordingly, the size of T-401 and the energy requirement increased. However, the same desired purity of FAME product was obtained using either thermodynamic/activity model. 4.1.5. Alkali removal Stream 303 (128 kg/h) was fed to neutralization reactor R-201 to remove sodium hydroxide by adding phosphoric acid (100% purity). The resulting Na3PO4 was removed in gravity separator X-302. When potassium hydroxide is used as an alkali catalyst, the resulting potassium phosphate may be used as a valuable byproduct (e.g., fertilizer). 4.1.6. Glycerine purification After removing the sodium hydroxide, stream 305 contained 85% glycerol. If a glycerine by-product with a higher grade (e.g., 92%) was preferred, this stream would pass to T-501 for further removal of water and methanol by distillation. When the UNIQUAC model was used, stream 305 contained 62% glycerol and was fed to T-501 to obtain 85% or 92% glycerine. Glycerine purification T-501 was designed with four theoretical stages and a reflux ratio of 2. Water and methanol were removed in distillate stream 501. At the bottom, 92% glycerine was obtained as a high quality by-product.

4.1.7. Waste treatment The compositions of streams 401A, 402 and 501 are listed in Fig. 2. Because of their small flows, these streams were treated as hazardous gas or liquid wastes. However, reusing these streams may be advantageous in the future, especially for larger scale processes. For example, stream 501 can be returned to T-301 as a washing solvent instead of fresh water. Recovery of the solid waste stream 306 from X-302 as a possible fertilizer credit is also feasible. Overall, these changes offer potential approaches for reducing waste treatment loads 4.2. Alkali-catalyzed process using waste cooking oil (process II) To lower the cost of biodiesel, a continuous alkali-catalyzed process from waste cooking oil was developed. In comparison to process I, a pretreatment unit, including esterification of the free fatty acids, glycerine washing and methanol recovery, was added. The pretreatment unit is shown in Fig. 3 while the remainder of the process is identical to that shown in Fig. 2. The characteristics of the main streams in process II are shown in Fig. 3. 4.2.1. Esterification The esterification reaction was carried out at 70 _C, 400 kPa and a 6:1 molar ratio of methanol to crude oil (Lepper and Friesenhagen, 1986). The fresh methanol stream 101 (128 kg/h), the recycled methanol stream 1111 (188 kg/h) and the H2SO4 stream 103 (10 kg/h) were mixed before being pumped into esterification reactor R-100 by pump P-101. The waste cooking oil stream 105A (1050 kg/h), containing 6% free fatty acids, was heated in exchanger E-100 to 60 _C before entering R-100. In R-100, all the free fatty acids were converted to methyl esters. After being cooled to 46 _C, stream 106 was forwarded to glycerine washing column T-100 to remove the sulfuric acid and water. 4.2.2. Glycerine washing The resulting water and acid catalyst (H2SO4) from R-100 must be removed completely before proceeding to the alkali-catalyzed transesterification. By adding 110 kg/h of glycerine at 25 _C and 200 kPa, all of the resulting water was removed from oil stream 110A after three theoretical stages of washing. Stream 110A from T-100 was sent to downstream transesterification unit R-101. On the other hand, stream 110B (336 kg/h) contained 60% unreacted methanol, 33% glycerol, 3% sulfuric acid, 3% oil, 1% water and traces of esters. Recovering most of the methanol in this stream for reuse in R-100 was a logical step, which was realized in methanol recovery column T-101. 4.2.3. Methanol recovery In T-101, five theoretical stages and a reflux ratio of 5 were used. At 28 _C and 20 kPa, 94% of the total methanol fed to the column was recovered in the distillate (i.e., stream 111) at the rate of 188 kg/h. It contained 99.94% methanol and 0.06% water and was recycled to R-100. At 70 _C and 30 kPa, bottom stream 112 (147 kg/h) was composed of 75% glycerol, 8% methanol, 7% sulfuric acid, 7% oil and 3% water. Due to the presence of sulfuric acid, this stream was not reused and was treated as waste. Nevertheless, neutralizing the sulfuric acid and then recovering the glycerol is a feasible alternative to reduce waste. Once the refined oil without free fatty acids is obtained, the downstream units are identical to those in process I using virgin vegetable oil. Compared to process I, despite the decrease in raw material cost by using waste oil, the addition of a pretreatment unit to reduce the content of free fatty acids in the feedstock oil in process II would be expected to more than offset these savings. 4.3. Acid-catalyzed process using waste cooking oil (process III) An acid-catalyzed system is insensitive to any free fatty acids in the oil. Consequently, process III, an acid-catalyzed continuous process from waste cooking oil (Fig. 4) appears to be a promising alternative to the alkali process. The description of process III concentrates on the differences from the previous processes I and II. 4.3.1. Transesterification

On the basis of discussions in Section 2.2, the reaction conditions were set to a 50:1 molar ratio of methanol to oil, a 1.3:1 molar ratio of sulfuric acid to waste oil, a reaction temperature of 80 _C and a pressure of 400 kPa. Fresh methanol (stream 101 at 216 kg/h), recycled methanol (stream 1201 at 1594 kg/h) and sulfuric acid (stream 103 at 150 kg/h) were mixed first and fed to transesterification reactor R101 by pump P-101. Waste cooking oil (stream 105 at 1030 kg/h) entered R-101 after being heated to 60 _C in exchanger E-101. In R- 101, 97% of the oil was assumed to be converted to FAME after 4 h. Two identical reactors operated in series were used, indicated as R-101A/B in Fig. 4. 4.3.2. Methanol recovery Because of the large excess of methanol in stream 106, methanol recovery was the first step following the reaction in order to reduce the load in the downstream units. In methanol distillation column T-201, five theoretical stages, a reflux ratio of 2 and vacuum distillation were employed. As in process I, a 94% methanol recovery rate was achieved in stream 201 and recycled to R-101. Bottom stream 202 was forwarded to acid removal unit R-201. 4.3.3. Acid removal For acid removal, the design principle was the same as for alkali removal in process I. In reactor R-201, sulfuric acid was completely removed in a neutralization reaction by adding calcium oxide (CaO) to produce CaSO4 and H2O. Calcium oxide was used primarily due to its low-cost relative to other alkali substances. Also the water produced would be absorbed by the resulting CaSO4 to form CaSO4_2H2O. However, since absorption of water by CaSO4 is relatively slow, in our current simulations CaSO4 rather than CaSO4_2H2O was considered as a solid waste. A gravity separator, X-201, was employed to remove the CaSO4. The resulting stream 203C (1247 kg/h) consisting of 79% FAME, 9% glycerol, 8% methanol, 2% unconverted oil and 2% water proceeded to water washing column T-301. In terms of equipment sizing and operating conditions, the remaining water washing column (T-301) and purification units (i.e., FAME purification column T-401 and glycerol purification column T-501) were similar to those used for process I. 4.4. Acid-catalyzed process using hexane extraction (process IV) To avoid the formation of emulsions due to water washing, the use of hexane or petroleum ether as a solvent following the procedures of Nye et al. (1983) and McBride (1999), to separate the FAME from other components was proposed in process IV (Fig. 5). The operating conditions for the units from reactor R-101 to methanol distillation T-201 were the same as those in process III. The following discussion pertains to the units downstream of methanol distillation column T-201. After T-201, the reaction mixture contained methanol, FAME, glycerol, sulfuric acid, unconverted oil. When hexane was added and a liquidliquid extraction was used in simulating hexane extraction of FAME, the results showed incomplete recovery of FAME in hexane. This was due partly to the lack of information in the HYSYS on interaction parameters in such a complex multi-component system and also to the fact that hexane is soluble in anhydrous methanol. To decrease hexane solubility, water was added to the methanol in the ratio 1:10 by volume, resulting a clear-cut separation of hexane/FAME and methanol/water phases (McBride, 1999). In addition, instead of a liquidliquid extraction unit, component splitters T-301A and T-301B were used in simulating the hexane extraction of FAME and the methanol/water washing in process IV. On the basis of our experimental results (McBride, 1999), we assumed that a good separation between the FAME/hexane and glycerol/methanol/water phases was achieved after the addition of hexane and water. Based on amounts of these components in feed streams 203 and 205, 100% of the hexane and the unconverted oil, 99.5% of the FAME, 4% of the methanol and the water, 2% of the sulfuric acid and 1% of the glycerol, were in the upper layer (stream 205A) from T-301A. The lower layer (stream 205B) consisted of the remaining glycerol, methanol, sulfuric acid and water. A second washing with methanol/water (T-301B) was applied to completely remove the water, glycerol and sulfuric acid from the hexane/FAME layer in stream 205A at room temperature. Detailed descriptions of these separations follow. 4.4.1. Hexane extraction

Based on the methanol volume in stream 203, an equal volume of hexane (0.13 m3/h) and 0.01 m3/h water (vol. ratio of water to methanol was 1:10) were added to T-301A at 25 _C and 200 kPa. After hexane extraction in T-301A, stream 205A was forwarded to a second washing unit (T-301B) at 25 _C and 150 kPa. The main components of bottom stream 205B were methanol (27%), glycerol (28%), sulfuric acid (40%) and FAME (1.4%). In T-301B, 0.14 m3/h methanolwater (80:20 v/v) was added. After T-301B, neither glycerol nor sulfuric acid remained in the FAME and hexane stream 301A. It was then passed to the downstream FAME distillation column T-401 to remove the hexane. Stream 205B from T301A was combined with stream 301B from T-301B. At a total flow rate of 492 kg/h, they entered R-201 for sulfuric acid removal by adding calcium oxide, as in process III. 4.4.2. FAME purification Because most of the methanol remained in the glycerol stream after the hexane extraction and second methanol/water washing, T-401 was principally used for distilling the hexane from the FAME. At five theoretical stages and a reflux ratio of 3, vent gases (stream 401A) were discharged from the top of T401 and recycled to T-301A to reduce the need for fresh solvent (stream 110). Distillate stream 401 was the FAME product (99.65%) and stream 402 from the bottom of T-401 contained any unconverted oil. 4.4.3. Glycerine purification Stream 303A entered T-501 for a five-theoretical stage distillation and a reflux ratio of 2. Glycerine with 85% or 92% purity, depending on the desired degree of purity, could be obtained in stream 502 by different energy requirements. A portion of top stream 501 (112 kg/h) was returned to T-301B as the solvent for the second washing.

KINETICS OF PALM OIL TRANSESTERIFICATION IN A BATCH REACTOR

D. Darnoko and Munir Cheryan Batch reaction. Transesterification reactions were performed in a 1-L three-necked flask equipped with a reflux condenser, a thermometer, and a sampling port. The reactor was immersed in a constanttemperature water bath equipped with a temperature controller, that was capable of maintaining the temperature within 0.2 C. Agitation was provided with a magnetic stirrer, which was set at a constant speed throughout the experiment. Initially, the reactor was filled with 500 g palm oil and heated to the desired temperature. A known amount of potassium hydroxide (the catalyst) was dissolved in the required amount of methanol and heated separately to the desired temperature. This methanolic KOH was then added to the base of the reactor (to prevent evaporation of methanol) at which point the reaction was assumed to have started. The molar ratio of methanol/oil was fixed at 6:1 since the literature suggests that this is the optimal ratio for vegetable oil transesterification. The weight of 1 mol oil was determined from the calculated average molecular weight of palm oil based on the known fatty acid composition of the oil. At various times, a sample was withdrawn quickly from the reactor with a Pasteur pipet. About 300 mg of the sample mixture was placed in a vial and diluted with 5 mL high-performance liquid chromatography grade tetrahydrofuran, and then one drop of 0.6 N hydrochloric acid was added to neutralize the catalyst. Separate studies had determined that this dilution and neutralization stopped the reaction immediately. The sample was then filtered through 0.2 m poly tetra fluoroethylene syringe filter and kept at -20/C until further analysis.

CINTICA DA TRANSESTERIFICAO DA OLENA DE PALMA AFRICANA COM ETANOL

Adrin vila, Antonio Bula e Homero Sanjun El reactor se carg con 500ml de aceite y se sumergi en bao de temperatura constante hasta alcanzar la temperatura deseada. Por separado se prepar etxido (solucin de etanol con KOH) y se calent

hasta llevarlo a la temperatura del aceite, se adicion al reactor y se comenz a medir el tiempo transcurrido. Se tomaron muestras a diferentes intervalos de tiempo durante el transcurso de la reaccin (120min), con una frecuencia mayor durante el comienzo de la reaccin. En total se tomaron nueve muestras, a los 0,5; 1; 1,833; 3; 5; 20; 35; 50 y 100min. Para detener la reaccin se utilizaron tubos de ensayo de 10ml que contenan 4ml de HCl 1N, cuya funcin es neutralizar Se tomaron muestras a diferentes intervalos de tiempo durante el transcurso de la reaccin (120min), con una frecuencia mayor durante el comienzo de la reaccin. En total se tomaron nueve muestras, a los 0,5; 1; 1,833; 3; 5; 20; 35; 50 y 100min. Para detener la reaccin se utilizaron tubos de ensayo de 10ml que contenan 4ml de HCl 1N, cuya funcin es neutralizar el catalizador y por tanto evitar la continuacin de la reaccin. En los tubos de ensayo se formaban dos capas, una acuosa donde residen las sales formadas por el cido y el catalizador, y otra capa donde estn los productos y subproductos de inters. De esta ltima capa de cada una de las muestras se tomaron 100l y se diluyeron en 10ml de 2-propanol. Una vez diluidas, las muestras se pasaron a travs de unos filtros de membrana de 0,45m y se almacenaron para su posterior anlisis. Los productos obtenidos en la reaccin de transesterificacin difieren de manera considerable en sus caractersticas fsicas y reactividad qumica, por lo cual se dificult conseguir un mtodo adecuado para realizar un mtodo nico de anlisis para las muestras tomadas. Despus de una extensa bsqueda se encontr que el mtodo ms adecuado, de acuerdo a los recursos disponibles y la viabilidad tcnica, era la cromatografa lquida de alta resolucin (HPLC). Se utiliz un cromatgrafo Lachrom-HITACHI Mod : L-7100, provisto de un detector UV, integrador, y una columna ZORBAX XDB C18. El flujo utilizado para la fase mvil fue de 1mlmin-1. Las curvas de calibracin se hicieron utilizando estndares para los etil steres, mono-di y triglicridos.

A SECOND ORDER KINETICS OF PALM OIL TRANSESTERIFICATION

Theerayut Leevijit, Worawut Wisutmethangoon, Gumpon Prateepchaikul, Charktir Tongurai and Michael Allen 2 .1 Materials A refined palm oil, a commercial methanol of 95 % purity, and a commercial sodium hydroxide pellets were used in this study. The free acid content of the oil was determined according to AOCS official method Ca 5a-40 [10] to be 0.64 %. Chloroform, hexane, diethyl ether, formic acid, and benzene used in the analysis were all of pro analysis grade. 2 .2 Apparatus The experimental set up is shown in Fig. 1. A sealed 2,000 mL glass reactor equipped with a mechanical stirrer, digital thermometer, and sampling port was used in this study. The reactor was immersed in a constant-temperature circulating water bath, Model EYELA SB-24 & T-80 (Tokyo Rikakikai Co.,Ltd, Japan); it was capable of controlling the temperature to within 0.1 C. A mechanical stirrer, Type R2R1 (Heidolph, Germanny), fitted with a stainless steel 6-blade disk turbine provided the sufficient mixing. The mixing intensity is presented by Reynolds number (NRe) of impeller in Eq. (1) [11].

where NRe is Reynolds number; n is turbine speed (rpm); Da is turbine diameter (mm); SG is fluid specific gravity; and is fluid dynamics viscosity (Pa.s). NRe was referred to the start of the reaction; it was around 2,000. During the reaction, samples were withdrawn with a 2 mL pipettes through the sampling port.

2 .3 Procedures The reactor was initially charged with 650 g of refined palm oil. The reactor assembly was then placed in the circulation water bath and heated to the designed temperature. A measured amount of methanol and sodium hydroxide solution was heated separately to the reaction temperature and added to the reactor. The mechanical stirrer was started as soon as possible. The reaction was timed when the solution was added. 2 .4 Sampling Samples of about 2 mL were withdrawn at pre-specified time intervals. Approximately 17 samples were collected during the reaction (90 min). At the beginning of the reaction, more frequent sampling was required. Each sample was collected in 20 mL test tube filled with 6 drops of 0.6 N sulfuric acid to neutralize the catalyst. The mixture was then suddenly washed twice with distilled water: first 8 mL of distilled water was added to the test tube and the mixture was washed in 300 mL flask filled with 200 mL of distilled water. Upon washing, glycerol, methanol and remaining sodium hydroxide or sulfuric acid were transferred to the water phase. At each time of neutralization and washing, a vigorous mixing was provided by the mixer. Separate studies had determined that these procedures stopped the reaction immediately. Then the ester phase and water phase formed separate liquid layers. The ester phase was then centrifuged to ensure a thorough separation. The ester phase was kept at 20 C until further analysis. 2 .5 Analysis The samples were analyzed by thin layer chromatography/flame ionization detector (TLC/FID) [12,13]. Analyses were performed using an Itronscan MK-6 with Chromarod type S-III quartz rod (Mitshubishi Kagaku Iatron Inc., Japan). The samples were diluted in chloroform and 1 L of the solution was spotted on each rod. The rods were developed in hexane/diethyl ether/formic acid (50:20:30 vol/vol/vol) for 8 cm and hexane/benzene (1:1 vol/vol) for 10 cm. The rods were dried and scanned under the following conditions: hydrogen flow rate 160 mL/min, air flow rate 2.0 L/min, speed 30 s/scan. ME, TG, DG, and MG were effective separated; peak areas were calculated with chromatography data system ChromStar. The weight percentages (%wt) of the reaction compositions on a glycerol-free basis were determined.

THE EFFECT OF WATER ON THE TRANSESTERIFICATION KINETICS OF COTTON SEED OIL WITH ETHANOL
Elena Bikou, Argiro Louloudi, and Nikos Papayannakos 2 Experimental 2.1 Materials

Taken into account only the fatty acids with a content higher than 0.5 wt.-%, its molecular weight is calculated equal to 860 [8]. Ethanol-water mixtures with 98.0 and 95.6% mole pure ethanol were prepared by properly mixing two types of commercial ethanol: i) absolute ethanol (99.4% mole ethanol) from Riedel de Haen and ii) 96% extra pure ethanol (87.5% mole ethanol) from Merck Co. Analytical grade potassium hydroxide pellets from Mallinckrodt Co. were used as catalyst in all cases. Experiments were carried out at 4:1, 6:1 and 9:1 ethanol/oil molar ratio and 99.4%, 98.0%, 95.6%and 87.5%mole ethanol in the mixture ethanol-water. 2.2 2.2 Transesterification Reaction Procedure 0.150 kg refined cottonseed oil and the appropriate quantity of ethanol-water mixture were added to a 500 ml three-necked flask equipped with a mechanical stirrer (vertical propeller, maximum speed 1500 rpm), thermometer and condenser. The mixture was stirred and heated up to boiling point, 78.2 0.2 _C in all cases, then the catalyst, 1 wt.-% (by weight of oil) potassium hydroxide, was added rapidly and timing of the reaction began. Heating was continued for 3 hours. Samples (3 ml each) were withdrawn at 2, 5, 10, 20, 30, 60, 90, 120 and 180 min reaction time and they were quickly quenched to stop reaction with cold (T = 24 _C ) deionized water. After being centrifuged, two phases were revealed: the upper transparent yellow oily phase consisting of the ethyl esters, the intermediates diglyceride and monoglyceride and the nonreacted triglyceride, and the lower water phase of white emulsion consisting of water, glycerin and minor contaminants (< 12 wt.-%). The residual catalyst and the nonreacted excess of ethanol were distributed between the two phases. The upper phase was purified with deionized water at a ratio of water/sample at least 3:1 vol/vol, for 3 times. Since the ethyl esters have an extremely high tendency to form an emulsion on contact with water, care was taken to gently agitate the samples while washing.

ACID-CATALYZED PRODUCTION OF BIODIESEL FROM WASTE FRYING OIL

S. Zheng, M. Kates, M.A. Dube, D.D. McLean 2.2. Experimental procedures 2.2.1. Apparatus The apparatus used for the transesterification reaction was a 5-L stainless-steel jacketed reactor, equipped with an internal cooling coil, a variable speed two-blade propeller turbine agitator, sampling port and a reflux condenser using cold tap water to condense methanol vapor (to prevent buildup of excessive pressure). The temperature of the reactor was controlled and maintained at 70 or 80 1C by means of LabViewTM software. 2.2.2. Procedure Methanol (reagent grade, ACP Chemicals Inc.), concentrated sulfuric acid (ACS grade, BDH Chemicals Inc.) and waste frying oil (from a local restaurant, Sams University Tavern, Ottawa) were used in the experiments. The waste oil (canola oil, derived from rapeseed oil (Brassica napus ssp. oleifera), free of erucic acid) contained 6% by weight of free fatty acid (FFA), 12% of diglyceride (DG), 82% triglyceride (TG) and little or no detectable monoglyceride (MG). It was used directly without further treatment. Standard triolein, diolein, monoolein, methyl oleate, oleic acid and glycerol were obtained from SigmaAldrich Co. (St. Louis, MO). Methanol and sulfuric acid were premixed in the mole ratios given in Table 1, and cooled to room temperature. Each mixture in turn was transferred to the reactor, which was then sealed and pressurized. The mechanical stirrer was started, the mixture was heated to the reaction temperature (70 or 80 1C), and the condenser cooling water was turned on. The appropriate amount of oil (see Table 1) was separately measured out, preheated to 70 1C and fed into the reactor by a liquid pump. The reaction was timed from when the oil was all pumped in (usually o1 min). Pressure was allowed to vary from 169 to 190 kPa (24.527.5 psia) at 70 1C and 266293 kPa (38.542.5 psia) at 80 1C. The reaction was allowed to proceed for 4 h or more. Five milliliter samples were collected at 5 min intervals for the first 30 min, and thereafter at 30 min intervals. All oil samples were freed from methanol, glycerol and sulfuric acid by partitioning between hexane and methanolwater (10:1 v/v), as described

elsewhere [9]. The hexane phase was separated and brought to dryness in vacuo in a rotary evaporator; the resulting oil was analyzed offline by GPC [9] for the oil to FAME conversion. The intermediate and final products (MG, DG, TG, and FAME) in the oil samples were also analyzed quantitatively by GPC, using calibration curves generated for pure standards of mono-, di-, and triolein and methyl oleate, respectively [9,10].

KINETICS OF TRANSESTERIFICATION OF SOYBEAN OIL

H. Noureddini and D. Zhu Materials. Refined and bleached soybean oils were obtained from Archer Daniels Midland Company (Lincoln, NE). Free acid content of the oil was determined according to AOCS method #Ca 5a-40 (15) to be 0.09%. Certified methyl alcohol of 99.9% purity was purchased from Baxter Diagnostics Inc. (McGraw Park, IL). Sodium hydroxide pellets of 98.2% purity were purchased from Fisher Scientific Company (Pittsburgh, PA). The high-performance liquid chromatography (HPLC) standards for methyl esters were obtained from Sigma Chemical Company (St. Louis, MO). A 0.907 wt% stock solution of sodium hydroxide in methanol was prepared and used in all of the reactions. A 6:1 molar ratio of this stock solution with soybean oil amounted to about 0.20 wt% sodium hydroxide in soybean oil. Reaction conditions. Experiments were designed to determine the reaction rate constants. A 6:1 molar ratio of alcohol to soybean oil, which is a typical ratio for Transesterification of vegetable oils, was used in all experiments. To examine the temperature dependency of the reaction rate constants, reactions at 30, 40, 50, 60, and 70C were studied. All reactions were carried out at atmospheric pressure. Three different mixing intensities were used to investigate the effect of mixing on the kinetic behavior. The rotational speed of the impeller was set at 150, 300, and 600 rpm. The mixing intensity may also be presented by Reynolds number (NRe). The impeller rotational speed and the NRe are related in Equation 1. where n is the rotational speed of the impeller, Da is the impeller diameter, and and are the fluid density and viscosity, respectively. The numerical values for NRe at the beginning of the reactions were determined to be 3100, 6200, and 12,400 for rotational speed of 150, 300, and 600, respectively. There is a significant increase in NRe values as the reaction progresses, which is primarily due to lower viscosity of the products. In the following sections when the mixing intensity is presented by NRe, it is referring to the start of the reaction. Apparatus. A 1500-mL glass cylindrical reactor equipped with a mechanical stirrer, thermometer, built-in condensing coil, and sample port was used in all kinetic experiments. The reactor was immersed in a constant-temperature circulating bath, Model 1157 VWR Scientific (Philadelphia, PA) which was capable of controlling the temperature to within 0.01C of the set point. A mechanical stirrer Model SL 1200, Fisher Scientific (Fairlawn, NJ) fitted with a stainless steel folding propeller provided the mixing requirements. During the course of the experiment, samples were withdrawn with a 2-mL disposable pipette through an opening on top of the reactor. Procedures. The reactor was initially charged with 200 g of soybean oil. The reactor assembly was then placed in the circulating constant-temperature bath and heated to the desired temperature. Measured amount of the methanol/sodium hydroxide stock solution (44.3 g), which was heated separately to the reaction temperature, was added to the reactor and the mechanical stirrer was started. The reaction was timed as soon as the mechanical stirrer was turned on. Sampling and analysis. Samples were drawn at pre-specified time intervals. Approximately 15 samples were collected during the course of each reaction (90 min). The frequency of sample collection varied and was dictated by the reaction conditions. Reactions at higher temperatures and mixing intensities required more frequent sampling at the beginning of the reaction. As the reaction temperature and mixing intensity were decreased, an initial delay in the reaction was experienced, and sample collection was delayed accordingly. This phenomenon is explained in later sections. Samples were taken at 1- and 2-

min intervals, early in the reaction, and at 10- to 15-min intervals, later in the reaction. Samples were collected in 10-mL test tubes filled with 4 mL of distilled water. The test tubes were kept in an ice bath at about 5C prior to use. Samples (2 mL) were quenched in the test tubes by placing them in the ice bath immediately after removal from the reactor. The test tubes were then shaken to stop the reaction. Upon washing, glycerol (GL) and sodium hydroxide are transferred to the water phase, and esters and unreacted GL form a separate liquid layer. The layers were then centrifuged at 1600 rpm for about 15 min to ensure a thorough separation. An HPLC (ISCO, Lincoln, NE), equipped with a model 2350 pump and a refractive index detector, Refracto Monitor. IV (Thermal Separation Products, Riviera Beach, FL), was used for the analysis. Temperature for the column was controlled with a circulating constanttemperature bath. Data collection and analysis were performed with Hewlett-Packard (Wilmington, DE) Chemstation Software. A Spherisorb OSD 2 column (Phase Separation Inc., Norwalk, CT) (250 4.6 mm with 80 pore size and 5 m particle size) was used for the separation. The HPLC mobile phase consisted of a 50:50 volumetric mixture of acetone and acetonitrile. The mobile system was degassed for 15 min in an ultrasonic bath before use in the HPLC system. The HPLC pump operated at 0.70 mL/min, and the column temperature was set at 35C. Standards for methyl esters, MG, DG, and TG were used to establish the calibration charts. Using these calibration charts, all the integration results were corrected for the weight percentage of the individual components.

INVESTIGATION OF THE REFINING STEP OF BIODIESEL PRODUCTION

Filiz Karaosmanoglu, K. Bars Cgzoglu, Melek Tuter, and Serap Ertekin Investigation of the Biodiesel Refining. The transesterification reaction scheme is shown in Figure 2. As can be seen, there are many components in the product mixture. First the ester product and then glycerin and byproducts must be obtained from this mixture. The three different methods used were (1) washing with hot distilled water, (2) dissolving in petroleum ether and then washing with distilled water, and (3) neutralization with sulfuric acid (1:1). The refining yield was calculated by dividing the amount of the pure ester product by the amount of the rapeseed oil used in the transesterification reaction. The water contents, acid values, and composition of the biodiesel obtained in these three different methods were investigated. All of the results are given as the average of the repeated experiment results. Refining Method of Washing with Hot Distilled Water. This method was applied by washing the mixture with distilled water at three different temperatures (50, 65, 80 C). After completion of the reaction, the mixture was cooled to room temperature and transferred to a separatory funnel by decantation. The catalyst was separated in solid state in the reaction vessel. Separation of the ester and glycerin phases was performed by letting them stand for 5 h in the separatory funnel. The crude ester phase was washed with hot distilled water at a 1:1 ratio in the following order: (1) two times without stirring (washings 1 and 2) and (2) three times with stirring (washings 3-5). Another operation was not applied to washing waters. The ester phase was left over the heated Na2SO4 (25% of the amount of the ester product) overnight and then filtered. Figure 3 shows the scheme of the hot water washing method. The refining yields and the results of the analysis for the determination of the water content, acid value, and composition of biodiesel at three different working temperatures are given in Table 3. The refining yield decreased with increasing temperature. At all three temperatures, the water amount, acid value, and ME composition of the ester product were found to be very similar.

Refining Method of Washing with Water and Petroleum Ether. After completion of the transesterification reaction, the mixture was cooled to room temperature and then transferred to a separatory funnel by decantation. The catalyst was separated in solid form in the reaction vessel. Following the separation of ester and glycerin layers, the methanol in the ester phase was removed in a rotary evaporator under vacuum. The ester phase was then taken again to the separatory funnel. After the addition of petroleum ether (equal to the ester phase volume) and distilled water (at 20 C and equal to twice the ester phase volume), the pH of the solution was adjusted to 7 by adding acetic acid. The washing process with distilled water was repeated three times. The ester phase was left over the heated Na2SO4 (25% of the amount of the ester product) overnight and then filtered. The petroleum ether was removed in the rotary evaporator under vacuum. The scheme of this method is shown in Figure 4. The results obtained with this method are shown in Table 4.

Refining Method of Neutralization with Sulfuric Acid (1:1). With the same principle, this method has been applied in two different ways by using solid catalyst and the solution of solid catalyst in methanol. The aim of using the solution of NaOH in methanol was the reobtainment of the catalyst as a salt resulting from neutralization with H2SO4. (a) Use of Catalyst in Solid Form. In this method, similar to the two previous methods, the catalyst was removed by decantation from the product mixture and the product mixture was transferred to a separatory funnel for separation of the ester and glycerin phases The pH of the ester phase was found to be 11.92. Sulfuric acid (1:1) was added until a neutral pH was obtained. The ester phase was separated by centrifugation from the salt formed by the addition of the acid. Methanol was removed in a rotary evaporator while water of the ester product was removed by leaving the product over the heated Na2SO4

(25% of the amount of the ester product) overnight. The scheme of this method is shown in Figure 5, and the results are given in Table 5.

(b) Use of Catalyst Dissolved in Methanol. The necessary amount of catalyst for the transesterification reaction (1.6% by weight of the oil) was dissolved in methanol and added to the reaction flask after heating to 68 C; the reaction was performed at 65 C. At the end of the reaction, the pH was found to be 13.07. The mixture was neutralized by adding H2SO4 (1:1) solution. After the removal of the salt (Na2SO4) formed by centrifugation, the ester phase was transferred to a separatory funnel, leading to separation of the glycerin and ester phases. The methanol in the ester phase was removed by a rotary evaporator, while the glycerin phase was removed by washing three times with distilled water (at 20 C). The water in the ester product was removed by leaving the product over the heated Na2SO4 (25% of the amount of the ester product) overnight and was then removed by filtration. The scheme of this method is shown in Figure 6, and the results are given in Table 6.