A two-step acid-catalyzedprocess for the production of biodiesel from rice bran oil

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Abstract :
A study was undertaken to examine the effect of temperature, moisture and storage time on the accumulation of free fatty acid in the rice bran. Rice bran stored at room temperature showed that most triacylglyceridewas hydrolyzed and free fatty acid (FFA) content was raised up to 76% in six months. A two-step acid-catalyzedmethanolysisprocess was employed for the efficient conver sion of rice bran oil into fatty acid methyl ester (FAME). The first step was carried out at 60 C. Depending on the initial FFA content of oil, 55–90% FAME content in the reaction product was obtained. More than 98% FFA and less than 35% of TG were reacted in 2 h. The organic phase of the first step reaction product was used as the substrate for a second acid-catalyzedmethanolysis at 100 C. By this two-step methanolysisreaction, more than 98% FAME in the product can be obtained in less than 8 h. Distillation of reaction product gave 99.8% FAME (biodiesel) with recovery of more than 96%. The residue contains enriched nutraceuticals such as c-oryzanol (16–18%), mixture of phytosterol, tocol and steryli ester (19–21%).

1. Introduction:
Biodiesel (BD) is receiving increased attention as an alternative, non-toxic, biodegradable, and renewable diesel fuel. iBD is usually produced by the transesterification of vegetable oil or animal fat with short chain alcohol. BD has higher oxygen content than petroleum diesel and its use in diesel engines have shown great reductions in emission of particulate matter, carbon monoxide, sulfur, polyaromatics, hydrocarbons, smoke and noise (Murayama, 1994). In addition, burning of vegetable-oil based fuel does not contribute to net atmo- spheric CO2 levels because such fuel is made from agri- cultural materials which are produced via photosynthetic carbon fixation. *

The source of BD usually depends on the crops amenable to the regional climate. In the United States, soybean oil is the most commonly BD feedstock, whereas in Europe, and in tropical countries the rapeseed (canola) oil and palm oil are the most common source for BD, respectively (Knothe, 2002). Perhaps the largest major impediment to wider commercialization of BD is its cost. When produced from refined edible oils, feedstock cost contributes more than 70% to the cost of BD (Haas et al., 2004). The use of inexpensive, non-edible feedstock and the utilization of by-products in the BD production may significa ntly reduce the cost of BD. Rice bran oil (RBO) offers significant potential as an alternative low-cost feedstock for BD production. Rice bran, a low valued co-product of rice milling, contains 15–23% RBO. Rice bran has great potential as a supplementary source of many nutrients. Recent findings show that rice bran is as good as or even better than oat bran in reducing serum cholesterol and reducing the risk of

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heart disease. However, immedia tely following the mill- ing process, rapid deterioration of the crude fat in the bran by lipase and, to a lesser extent, oxidase makes the bran unfit for human consumption. Rice bran con- tains several types of lipase that are site specific and cleave the 1,3-site of triacylglycerol (TG). Depending on the nature of bran and the storage conditions, spoil- age due to lipase continues after the milling (Takano, 1993). Rapid increase in the free fatty acid (FFA) con- tent in the rice bran occurs within hours, followed by about 5% per day increase in FFA content. The produc- ing of an offflavor and soapy taste, and the change of functional properties of bran were also reported (Ram- ezanzadeh et al., 1999). Heating of bran immedia tely after milling inactivate the lipase and prohibit the for- mation of FFA. Various methods for the stabilization of rice bran have been describedin the past (Lakkakula et al., 2004). However, due to the dispersed nature of rice milling, it is difficult to collect bran continuously from many mills in large quantities, thus making central treatment impractical. Moreover, stabilization of bran results in additional cost. As a result, the utilization of rice bran is limited and is mainly used as animal feed and boiler fuel (Goffman et al., 2003; Shih et al., 1999). Hexane is commonly used as the solvent in the commerciallyextraction of oil from rice bran. RBO is one of the most nutritious oils due to its favorable fatty acid composition and a unique combination of naturally occurring biologically active and antioxidant compounds, such as c-oryzanol, vitamin E, phytosterols, and tocotrienols (Cicero and Gaddi, 2001). In addition, rice bran also contains high molecular weight wax esters, which is a source of policosanols (Cravotto et al., 2004). Although RBO is highly nutritional, it is not popular worldwide and its production is limited by several fac- tors. Crude RBO has been difficult to refine because of its high content of FFA, unsaponifiable matter and dark color (Bhattacharyya et al., 1983). The refining loss for RBO is particular acute because it is 2–3 times the per- centage of FFA content in the oil. Due to rapid splitting of lipid by active lipase present in the bran, RBO avail- able in most Asian countries contains 40–50% FFA (Kosugi et al., 1994). Crude RBO with less than 5% FFA is desirable for economic refining purposes (Eno- chian et al., 1980). This leads to lack of widespreadcom- mercial use of RBO due to economic factors (Hegsted and Kousik, 1994; McCaskill and Zhang, 1999). As a re- sult, only a small portion (<10%) of RBO is processed into edible oil. Hence RBO with high FFA content is a potential cheap feedstock for BD production. After the removal of methylesters (ME) fraction in the product gives a lipid fraction that contains nutraceu- tical, biologically active, and antioxidant compounds such as coryzanol, tocopherols, tocotrienols phytoster- ols, polyphenols and squalene. Separation and purifica- tion of these compounds further reduces the cost of BD

and make it competitive with that of petrol–diesel. Data on the suitability of rice bran oil with varying FFA con- tent for the production of BD with fewest adverse effects on the functionality of biologically active by-product have not been reported in the literature. The first objective of this investigation is to understand the effect of temperature, moisture and storage time on the free fatty acid content in storage bran. The second objective is to develop an efficient method for the production of biodiesel from rice bran oil with varying FFA content with the fewest adverse effects on the functionality of biologically active by-product.

2. Methods 2.1. Materials
Rice bran was donated by a rice mill located in Taoyung County, Taiwan. Refined RBO, soybean oil, sunflower oil, rapeseed oil, palm oil, cottonseed oil, and coconut oil were purchased from Ushodaya Co. Ltd. (Hyderabad, India). Methanol (HPLC grade) and sulfuric acid (ACS grade, 95–97%), were purchased from Ac- ros Organics (Geel, Belgium). Standard c-oryzanol was kindly donated by Tsuno Rice Fine Chemicals Co. Ltd. (Wakayama, Japan). Standard tocopherol, sterol and other lipid sample were obtained from Sigma Chemicals Company (St Louis, MO). All solvents and reagents were either of HPLC grade or AR grade. All other chemicals used were obtained from commercial sources.

2.2. Storage condi ions of rice t bran
Bran was passed through a mesh (<0.6 mm) to remove foreign material, put in sealed polyethylene bags (250 g each) and stored at different temperatures (70, 50, 25, and 5 C) for up to six months to examine effect of temperature on FFA content in the bran. To study the effect of moisture content on FFA formation in the bran, four samples with different moisture levels were prepared from rice bran with initial moisture con- tent of 10.3% (control sample). The moisture content of the bran was determined by the AOCS official method (Official AOCS, 1997, Method Bc 2-49). A dried sample was prepared by heating the control rice bran under vacuum using rotary evaporator at 95 C for about 1 h. Two other samples were prepared by spraying 10 and 20 wt% of water to the control sample. Each sample with specific moisture content was mixed thoroughly and stored at the above-mentioned temperatures.

2.3. Extraction of RBO
Crude RBO was extracted from rice bran (50 g) by Soxhlet extraction for about 3 h, using hexane as the sol-

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vent. Dewaxing/deg umming of crude RBO was carried out as described by Kaimal et al. (2002). Preparation of fatty acid (FA) from crude RBO and other oils by saponification were carried out as described elsewhere(Vali et al., 2003).

2.4. Acid-c atalyzed RBO oil

methanolysis of

2 wt% sulfuric acid and nine times the stoichiometric amount of methanol required for the total conversion of TG. The reaction vessel was closed securely and sealed with Teflon tape. The methanolysis was per- formed under stirring (300 rpm) at 100 C by immers the vessel ing in a preheated oil bath.

2.6. Distillation FAME

of

Acid-catalyzed methanolysis reactions were carried out with 10 g of dewaxed/degu mmed RBO at 60 C and atmospheric pressure under stirring at 300 rpm. The average molecular weight of RBO is calculated based on its FFA and TG content, assuming a molecu- lar weight 870 for TG and 282 for FFA. Methanolysis reactions were performed in a 50 mL three-necked roundbottomed flask equipped with a reflux condenser, thermometer and rubber septum and placed in an oil bath with a temperature controller and magnetic stirrer. Dewaxed/de gummed RBO (10 g) were initially heated to 100 C for 20 min under vacuum (40 mm Hg) to remove trace water. Sulfuric acid (1–5 wt%) dissolved in metha- nol was injected into the reaction vessel through a sep- tum after the RBO were heated to the required temperature (60 C) under stirring. Samples (200 lL) were withdrawn at regular intervals and the water solu- ble components were removed by water washing (30.5 mL). The organic phase was dissolved in diethyl ether and analyzed by high temperature gas chromato- graph (HTGC) after drying over sodium sulfate. The es- ter conversion was estimated based on change in the percentage composition of TG, FFA, FAME, and par- tialglycerides before and after the reaction. The contents of other minor component in RBO were neglected in the calculation of conversion.

2.5. Two-st p acid-cat lyzed methanolysis of e a RBO
A two-step acid-catalyzed methanolysis of RBO was adopted for the complete conversion of FFA and TG to fatty acid methyl esters. The first step was carried out at 60 C and atmospheric pressure. The molar ratio of methanol/FFA was 5/1. Typically, 100 g dewaxed/degummed RBO was put into a two-necked 250 mL round bottomed flask equipped with a reflux condenser and rubber septum. The flask was immersed in an oil bath with a temperature controller and magnetic stirrer pre- heated to required temperature. Sulfuric acid (2 wt%) was mixed with required amount of methanol and trans- ferred into the reaction medium with syringe. Samples were withdrawn in every 10 min and its composition was analyzed by HTGC. After 2 h the contents were cooled to room temperature, and reaction product was washed with water (350 mL). The organic phase was col- lected and dried under vacuum (40 mm Hg) at 100 C for 20 min. The dried sample (50 g) was put in a 100 mL Schott Duran bottle (West Germany), mixed with

The distillations were performed in a 250 mL three necked round bottomed flask without stripping medium. The equipment includes a temperature controller, a re- ceiver flask connected to a vacuum sensor and a con- denser. A pump connected to the condenser provided vacuum. Product from the methanolysis reaction (100 g) was fed into the flask, the vacuum was adjusted to 5 ± 1 mm Hg. The temperature was first raised to 160 C for about 1 h; the distillate collected was referred as the first fraction. The temperature was then raised to 200 C in 30 min; the distillate collected was referred as the second fraction. Finally, the temperature was raised to 220 C in 20 min and the distillate was collected. The distillation was terminated when no more distillate appeared. The resulting residue (distilland) in the distilla- tion flask was cooled to ambient temperature before releasing the vacuum. The weightsof feed, residue (dis- tilland) and three fractions of distillate were determined and their contents of FAME, FFA, and acylgycerides were analyzed by HTGC. All analyses were performed with three replications. The AOCS methods were adopted for the determination of acid value of FFA (Official AOCS, 1997, Method, Ca 5a40). c-Oryzanol content was determined spectrophotometrica lly by mea- suring ultraviolet (UV) absorption at 314 nm in a 1-cm cell of solution in hexane followed by calculation using the specific extinction coefficient of 358.9 (Seetharama- iah and Prabhakar, 1986).

2.7. Analysis of reaction product
The fatty acid composition of saponified oil was determined after converting into their corresponding FAME by heating with BF3-methanol. The fatty acid composition was analyzed using authentic standards by gas chromatography as described elsewhere (Vali et al., 2003). The composition of FAME, FFA and acylglycerols in the reaction mixture and phytosterol, steryl esters and tocol content in the residue were determined by HTGC. Chromatographic analysis was performed on a Shimadzu GC-17A (Kyoto, Japan) gas chromato- graph equipped with a flame ionization detection system. Separations were carried out on a DB-5HT (5%-Phenyl)methylpolysiloxane nonpolar column (15 m · 0.32 mm; Agilent Tech. Palo Alto, California). The operating conditions were the following: The temperatures of injector and detector were set at 380 C.

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FFA & TG content (wt%)

The temperature of the column was held at 80 C for 0.2 min, increased at 15 C /min up to 380 C and then maintained at 380 C for 10 min. The split ratio was 1:50 and carrier gas used was nitrogen. Samples of 10 mg were dissolvedin 1 mL chloroform and 1 lL sample was injected into HTGC. Peaks in the chromatogram were identified by comparing retention times with standards of known compositions. Peaks less than detectable’’ (ND).

90 80 70 60 50 40

20 10 0 0 4 8 12 16

RB 1 (FFA) RB 1 (TG) RB 2 (FFA) RB 3 (FFA)

3. Results and discussion 3.1. Characteristic of rice bran samples
Table 1 shows some characteristics of three rice bran samples. The moisture content and the oil content shown in the table are typical values of commercial rice bran. The major lipids in fresh rice bran are neutral lip- ids (16–20%), of which 80–90% is TG (Aibara et al., 1986). The quality of bran depends on nature, degree of milling and age of rice grain and it can be determined in terms of FFA content in the oil. Storage time (weeks)

20

24

Fig. 1. Effects of storage time on the hydrolysis of triglycerdes (TG) and the formation of FFA in stored rice i bran. The samples were stored at ambient temperature (25 ± 2 C) for six months. FFA: free fatty acids; TG: triglycerdes; RB: rice bran. i

3.2. Effect of storage time on fatty acid content
Rancidity caused by the presence of active lipase in milled bran makes it unsuitable for human consump- tion. To better understand this lipolytic process, three rice bran samples with different initial FFA contents were stored at 25 ± 2 C for up to six months. The time-courses of changes in FFA and TG contents of stored rice bran are presented in Fig. 1. Hydrolysis of TG was continued in all samples regardless of their ini- tial FFA contents (Table 1). The FFA content increased steadily throughout the storage period. The fatty acid contents increased rapidly in the first six weeks, then in- creased slowly and leveled off at about 24 weeks. The trend in the decreasing of TG content is similar to that of fatty acid as can be seen from Fig. 1. Thus, rapid increasing of FFA in rice bran was observed in the first six weeks of storage.

3.3. Effect of moistu level on FFA re formation
The effect of moisture content in the rice bran on the formation of FFA is shown Fig. 2. Rice bran samples with varying moisture content were stored at ambient temperature (25 ± 2 C) and their FFA content were determined over a period of 24 weeks. Oils extracted from the control sample and from sample with 10 wt% added water contain more FFA than rice bran with high moisture (20%). Inactivation of lipase was confirmed for fresh rice bran dried at 95 C under vacuum for 1 h. FFA content of dried bran increases only slightly in the first week then remains fairly constant thereafter.

3.4. Effect tempera ture

of

storage

Fig. 3 shows the effect of temperature on FFA accumulation in stored rice bran. The results show that rice bran lipase is more active at 30 and 50 C than at 5 and 70 C. FFA content in the RBO showed a steady increase throughout the storage and the FFA content rose from 6.6% to 76% and 82% in 24 weeks when the bran was stored at 30 and 50 C, respectively.

Table 1 Some characteristic of rice bran samplesa,b Rice bran Characteristic of rice bran samples (%) Oil FFA TG Partialglyceridesc Moistur 10.5 ±e0.4 18.7 ± 0.3 6.6 ± 0.22 85.2 ± 0.8 8.2 ± 0.3 RBRB10.8 ± 0.3 18.2 ± 0.4 16.8 ± 0.36 78.4 ± 0.5 9.2 ± 0.8 II RB11.5 ± 0.3 16.3 ± 0.4 24.5 ± 0.41 60.8 ± 0.4 14.7 ± 0.4 III a Oil was extracted by Soxhlet extraction for 3 h with hexane. b Crude RBO was subjected to dewaxing /degummingbefore analyzing for FFA, TG and partialgl cerides by HTGC. y c Monoglyceri e and diglyceride. d

80 70
FFA content (wt%)

60 50 40 10.3%) 30 20 10 0 0 4 8 12 16 20 24 Storage time (weeks) Drie d Control (moisture Control+10% water Control+20% water

bean oil and sunflower oil undergo rapid oxidation and polymerization (Dunn, 2002). In contrast, the S/U value of dewaxed/degu mmed RBO is intermediate and therefore is suitable as feedstock for BD production without further refining. RBO with high FFA content is unsuitable for processing into edible oil. However, it is potentially a cheap feedstock for biodiesel production. Recently, there are numerous researches on producing biodiesel from vegetable oils. Table 2 lists the fatty acid composition of crude RBO, dewaxed/degu med RBO, and several vegetable oils m commonly employed in stud- ies to produce biodiesel.

3.6. Methanolysis of RBO
Alkali-catalyzed transesterification is used in the commercial production of BD. At atmospheric pressure and temperatures between 40–65 C, the base-catalyzed reaction can reach 95 wt% conversion in 1 h (Freedman and Pryde, 1982; Kusy, 1982). However,for alkali-cata-

Fig. 2. Effect of moistureon FFA formation in stored rice bran (RB1). Initial FFA content of rice bran: 6.6%. RB samples with different moisture contents were prepared as described in Section 2.2. The samples were stored at ambient temperat re (25 ± 2 C) for 24 weeks. u 80
FFA content (wt%)

5 ºC 30 ºC 50 ºC 70 ºC

fats) must be dried (moisture level <0.06%) and free of FFA (<0.5%). Freedman and Pryde (1982) reported that ester yields were significantly reduced if the reactants did not meet these requirements. The presence of minor amount of FFA and moisture in the reaction mixture produces soap, which lowers the yield of esters and rening difficult. FFA also consumed the catalyst and reduced catalyst efficiency. Thus, highly refined vegeta- ble oils are required. RBO contains 6–70% FFA depend- ing on the quality of rice bran. It is unsuitable as feedstock oil for the production of BD by alkali-catalyzed reaction. The alternative is the acid catalyzed reac- tion. Although acid catalyzed reaction is much slower, it does have advantages such as reduced purification costs and the ability to utilize less expens low-grade feed- stock (Goff ive et al., 2004). Fig. 4 shows the results of acid-catalyzed methanolysis of dewaxed/deu mmed RBO with varying FFA contents at atmospheric pres- sure and 60 C using 1:10 molar ratio of oil/methanol and 2 wt % of sulfuric acid as catalyst. It can be seen that the rate of methanolysis and the final methyl ester con- tent in the product depend on the initial FFA content in the RBO. About 96% ME content in the product can be obtained in 8 h for RBO with initial FFA content of 76%. As shown in Fig. 4, rapid formation of FAME was observed in the first 2 h due to rapid methanolysis of FFA. Analysis of reaction products showed an acid value of 2–3 wt% and it remained the same even after 24 h of reaction. This residual acid value might be due to the presenceof phenolic compounds such as oryzanol and tocols in the RBO. Moreover, the presence of other minor components such as phospholipids, oxidized lipids and amino acids, also contributes to the acidity

6 0 5 0 4 0 3 0 2 0 1 0 4 12 16 24 Storage time (weeks) 8 20

Fig. 3. The effect of storage temperature on formation FFA in stored rice bran (RB1). Initial FFA content of bran: 6.6%; Moisture content 10.3 wt%.

3.5. Fatty acid composi of RBO and other tion edible oils
Crude RBO contains 3–4% wax esters and 1–2% phospholipids. The high saturated to unsaturated fatty acid ratio (S/U) of crude RBO is attributed to the pres- ence of high molecular weight saturated wax esters and other mucilaginous compounds. Simultaneous dewaxing/degumming removes most wax esters (>90%), phospholipids (>95%) and other mucilaginous matter (Kaimal et al., 2002). The fatty acid composition of dewaxed/degu mmed RBO is similar to that of other re- fined oils, which are commonly used as feedstock in BD production. For oils with high S/U value such as palm oil, coconut oil and cottonseed oil, winterization is needed before such oils are used as BD feedstock. On the other hand, BD feedstock with lower S/U and high content of polyunsaturated fatty acid such as soy-

Table 2 Typical fatty acid compositiona (%) of crude RBO, dewaxed/degmmed RBO and other u refined vegetable oils Fatty acid CRBOb D/DRBOc Soybean Sunflower Cottonsee
Lauric 0.2 ± 0.1 n.d.
d

Myristic 0.8 ± 0.1 0.21 ± 0.1 n.d. n.d. Palmitic 17.7 ± 0.53 14.7 ± 0.47 10.4 ± 0.52 6.06 ± 0.25 Palmitoleic 0.23 ± 0.1 0.26 ± 0.1 0.35 ± 0.1 n.d. Stearic 2.2 ± 0.15 1.86 ± 0.22 4.7 ± 0.35 4.8 Oleic 40.6 ± 0.77 42.2 ± 0.68 24.8 ± 0.66 20.5 ± 0.63 Linoleic 35.6 ± 0.56 37.8 ± 0.51 52.5 ± 1.52 67.7 ± 1.2 Linolenic 1.8 ± 0.22 2.39 ± 0.1 6.5 ± 0.33 0.4 ± 0.11 Arachidic 0.2 ± 0.1 n.d. 0.32 ± 0.1 0.31 ± 0.1 Behenic 0.3 ± 0.1 0.2 ± 0.1 0.22 ± 0.1 n.d. Lignoceric 0.6 ± 0.26 0.3 ± 0.14 0.21 ± 0.1 0.23 ± 0.1 Saturated/unsaturated 22/78 = 0.282 17.31/82.69 = 0.206 15.85/84.15 = 0.188 0.3685 46.28/53.72 = 0.8615 a Mean absolute deviations of three indepen dent determinations. b CRBO: crude rice bran oil. c D/D RBO: simultane usly dewaxed o /degummedrice bran oil. d Peak areas less than 0.2% were considered to be negligibleand labeled as ‘‘not detectable’’ (ND).

n.d.

n.d.

Palm 0.2 ± 0.1 1.2 ± 0.05 1.11 ± 0.2 20.6 ± 1.2 40.20 ± 1.42 0.8 ± 0.11 0.42 ± 0.1 4.6 ± 0.31 4.5 ± 0.35 19.54 ± 0.43 43.3 ± 1.33 52.5 ± 1.44 9.0 ± 0.37 0.23 ± 0.05 1.0 ± 0.23 0.31 ± 0.11 0.27 ± 0.14 0.22 ± 0.08 n.d. n.d. n.d. 11.4/88.6 = 0.128 26.93/73.07 =
d n.d.

10 0 9 0 8 0 7 0 6 0
Methyl ester content (wt%)

2001; Kawahara and Ono, 1979). Our results with acid catalyzed methanolysis of RBO show that there is still considerable amount of fatty acid (1–2.5%) left after 24 h of reaction. It is necessary to decrease the FFA content to less than 0.5 wt% if the product of acid-catalyzed reaction is to be used as substrate for base-catalyzed RBO (49.4% FFA) RBO (75.8% FFA) RBO (63.3% FFA) RBO (6.6% FFA) RBO (24.5% FFA) 10 12 14 16 22 24 26 Reaction time (h) 18 20 amount of fatty acid in the product of acid-catalyzed methanolysis of high FFA feedstock and the need of multiple pretreatments before the product of acid-catalyzed reaction can be used as substrate for base-catalyzed reactions (Hammond, 1998; Canakci and Van Gerpen, 2001). However, if base catalyzed alcoholysis of RBO is used, the bioactive compounds such as oryz- anol and tocol will be lost since they are phenolic com-

5 0 4 0 3 0 2 0 1 0

2

4

6

8

0 Fig. 4. Time-course of acid-catalyzed methanolysis of rice bran oil with varying FFA contents. Reaction conditions: Dewaxed/ egummed d rice bran oil (10 g) was subjected to methanolysis reaction at atmospheric pressure and 60 C using 1:10 molar ratio oil/methanol and 2 wt% sulfuric acid as catalyst. RBO: rice bran oil; FFA: free fatty acids .

well known that acid-catalyzed alcoholysis of triglycerides is a slow reaction. Substantial amount of unreacted triglyceride were detected (data not shown) even after 24 h if the starting oil contains more than 20% FFA. Further increase in reaction time or methanol concentration had negligible effect on the transesterification of TG. Our results are in agreement with the results published by earlier workers (Canakci and Van Gerpen, 1999; Jeromin et al., 1987). An two-step alcoholysis (acid-catlyzed followed by base-catalyzed) has been developed by earlier workers for using oil with high FFA content as feedstock for the production of BD (Canakci and Van Gerpen,

of oil during alkali titration (Zhou and Ackman, 1996). It is

pounds and are susceptible to base treatment (Seetharamaiah and Prabhakar, 1986). Recently Goff et al. (2004) reported a single step acidcatalyzed alcoholyis of soybean oil in a sealed reactor. The advantage of this process is that feedstock oil with 1–2% FFA can also be alcoholysed effectively. After 26 h, methyl ester content higher than 99% in the product was reported. When dewaxed/degu med RBO with m 24.5% and 49.5% FFA contents were used as the substrates in the acid-catalyzed methanolyses at 100 C, the FAME content in the product are 62% and 73%, respectively. Increase in the methanol concentration or catalyst amount had negligible effect on the conversion. Water generated during the methanolysis of RBO with high FFA content formed another phase which absorb methanol and sulfuric acid. This water phase probably prohibited the transesterification of TG and further esterification of residual FFA. Substantial reduction in the conversion during acid-catalyzed alcoholysis of vegetable oil with excesse water (>1 wt%) has been reported (Goff et al., 2004; Canakci and Van Gerpen, 2001).

3.7. Two-step acid-catalyzed methanolysis of RBO
A two-step acid-catalyzed methanolysis process was adopted for obtaining high conversion in reasonably short time, using RBO with high FFA (>20%) as feed- stock. The first step was performed at 60 C using 2 wt% sulfuric acid and an oil/methanol molar ratio of 1:5. At a reaction time of 2 h, when more than 98% FFA were converted into their corresponding ME, the organic phase in the reaction product was removed as described in the experimental procedure and used as the substrate for the second methanolysis reaction at 100 C. Fig. 5 shows the time-course of variation of product composition in the two-step acid catalyzed methanolysis of RBO with an initial FFA content of 49.8%. The vertical dotted line indicates the end of the first step, and the beginning of the second step reaction. At the end of the first step, the reaction product contains 62% FAME, 3.2% FFA and 34.8% acylglycer des. i Mehtanolyssis of this product at 100 C resulted in more than 96% FAME content in the final product for a total reaction time of 8 h. Thus through this two-step acidcatalyzed methanolysis reaction, efficient conversion of low grade RBO into FAME can be accomplished in reasonably short time.

large number of patents and papers have been cited on the potential applications of these byproducts in cos- metic, pharmaceutical, food, polymer, and leather industries. Success in the purification and isolation of these byproducts could address the economic barriers to a wider adoption of biodiesel fuel. In this study, var- ious samples of crude RBO were subjected to simulta- neous dewaxing/degumming which gave crude rice bran wax (3– 4 wt%) and clear RBO. An efficient method was developed for the preparation of food grade rice bran wax from crude rice bran in this laboratory. The purified wax meets the USFDA specifications and can be used in cosmetic formulations. In addition, rice bran wax is also a rich source of high-molecular weight ali- phatic alcohols known as policosanol. Earlier investiga- tors have demonstrated the multitude of beneficial therapeutic properties associated with policosanol in- take. Policosanol was also used in different pharmaceu- tical formulations for the treatment of gastric and duodenal ulcers (Hernandez et al., 2002; Cravotto et al., 2004). Two-step acidcatalyzed methanolysis of RBO followed by distillation gave FAME (99% pure) with 96% recovery as distillate and a black brown colored residue (distilland). RBO sample contains 1.4– 1.8% c-oryzanol, 520–650 ppm vitamin E (tocopherols and tocotrienols), 1.6–2.2% phytosterols and sterylest- ers. Distillation of FAME at 5 mm of Hg vacuum and 220 C enriched these compounds in the residue. Depending on their initial contents and distillation conditions, the residues contains 16–18.2% c-oryzanol, and 19–21% mixture of sterols, sterylesters and tocol. Furthermore, defatted rice bran is a rich source of proteins (16–20%) (Landers and Hamaker, 1994) ˜ and phytochemicals such as phytic acid (5–6%) (Garco˜ N a-Estepa

The use of RBO as raw material for the production of biodiesel generates by-products such as defatted rice bran, wax ester, c-oryzanol, phytosterol and tocol. A
---------------------------------------------

1st 10 Step 0 9 0 8 0 7 0
Components (wt%)

2nd Step

et al., 1999) and myo-inositol. In the past few years, numerous in vitro and in vivo studies have shown that phytic acid and its isomers exhibit anticancer, antiinflammatory effects and has also beneficial effects such as protection against heart diseases, diabetes and renal calculi (Chen and Li, 2003). Purification and isolation of the antioxidant and biologically active compounds are currently under investigation in this laboratory.

FFA Acylglycero ls FAME

6 0 5 0 40 30 20 10 0 0 1

Acknowledgement The financial support of National Science Council of Taiwan under grant NSC 93-ET-7-011-001-ET is grate2 3 4 5 6 7 Reaction time (h) 8 9 10 11 fully acknowledged.

Fig. 5. Time courses of product composition during twostep acid catalyzed methanolysis of RBO (initial FFA content = 49.8%). The vertical dotted line indicates the end of the first step reaction. Reaction conditions were the same as those described in the experimental procedure. FFA: free fatty acids, Acylglyce rols: mixture of triacylglycerols, diacylglycerols and monoacylglycerols, FAME: fatty acid methyl ester.

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