J Am Oil Chem Soc DOI 10.1007/s11746-015-2634-3

ORIGINAL PAPER

Methyl Esters (Biodiesel) from and Fatty Acid Profile of Gliricidia sepium Seed Oil Gerhard Knothe1 · Maria Ellenita G. de Castro2 · Luis F. Razon3 

Received: 15 January 2015 / Revised: 6 March 2015 / Accepted: 6 March 2015 © AOCS (outside the USA) 2015

Abstract  Increasing the supply of biodiesel by defining and developing additional feedstocks is important to overcome the still limited amounts available of this alternative fuel. In this connection, the methyl esters of the seed oil of Gliricidia sepium were synthesized and the significant fuelrelated properties were determined. The fatty acid profile was also determined with saturated fatty acids comprising slightly more than 35 %, 16.5 % palmitic, 14.5 % stearic, as well as lesser amounts of even longer-chain fatty acids. Linoleic acid is the most prominent acid at about 49 %. Corresponding to the high content of saturated fatty acid methyl esters, cold flow is the most problematic property as shown by a high cloud point of slightly >20 °C. Otherwise, the properties of G. sepium methyl esters are acceptable for biodiesel use when comparing them to specifications in biodiesel standards but the problematic cold flow properties would need to be observed. The 1H- and 13C-NMR spectra of G. sepium methyl esters are reported.

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. * Gerhard Knothe [email protected] 1

Agricultural Research Service, National Center for Agricultural Utilization Research, US Department of Agriculture, 1815 N. University St., Peoria, IL 61604, USA

2

Biology Department, De La Salle University, Manila, The Philippines

3

Department of Chemical Engineering, De La Salle University, Manila, The Philippines





Keywords  Biodiesel · Fatty acid profile · Fuel properties · Gliricidia sepium seed oil · Legume · Methyl esters · Transesterification

Introduction With the interest in and use of alternative fuels derived from renewable biological sources continuing to grow, the need to increase their supply has caused an intense search for additional feedstocks. Biodiesel [1, 2], defined as the mono-alkyl esters of vegetable oils, animal fats or other materials composed largely of triacylglycerols and ranking among the prime alternative biofuels, is affected by this issue. The current supply of biodiesel suffices to replace only a low percentage of the petrodiesel market. Besides the supply issue, the search for additional biodiesel feedstocks has been powered by the perceived food vs fuel issue and economics. Thus, besides common commodity vegetable oils such as palm, rapeseed (canola) and soybean, numerous other potential feedstocks have been investigated. Some “alternative” vegetable oils or other feedstocks that have been studied include camelina [3], coriander [4], cuphea [5], desert date [6] jatropha [7], mahua [8], moringa [9], mustard [10], pennycress [11], pongamia [12], pumpkin seed [13], rubber seed [14], Thespesia populnea [15], and, especially under economic aspects, used cooking oils [16, 17] as well as, prominently in recent years, algae [18, 19]. Methyl esters, obtained by transesterification of the triacylglycerol-containing feedstock in the presence of a base catalyst, preferably alkoxide, are the most common form of biodiesel because methanol is in most countries the least expensive alcohol. Thus, biodiesel standards are largely based on the methyl esters. The most commonly

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applied biodiesel standards are ASTM D6751 [20] in the United States and EN 14214 [21] in Europe. Advantages of biodiesel are lower regulated exhaust emissions with nitrogen oxides (NOx) being an exception, inherent lubricity, high flash point, low or no sulfur content, and a positive energy balance besides constituting a renewable domestic feedstock. On the other hand, technical issues besetting biodiesel are poor cold flow properties and low oxidative stability, increased NOx exhaust emissions although this issue may fade with time due to increased use of exhaust emissions control technologies besides the aforementioned aspects of economics and supply. The fuel properties are, to a large degree, influenced by the fatty ester composition of the biodiesel as the various feedstocks yield biodiesel with fatty esters of varying chain length and unsaturation and, therefore, properties. Gliricidia sepium [22–26], also known popularly as madre de cacao or kakawate (in the Philippines) or by other names in different locations around the world, is a small to medium-sized tree attaining 10–12 m height. G. sepium is leguminous and thus nitrogen-fixing, not requiring nitrogen fertilizers [26]. It belongs to the Robinieae tribe of the Fabaceae family and is native to Central America but has been exported to other parts of the world including the Caribbean, the Philippines, India, Sri Lanka, West Africa and in the United States the southern tip of Florida [22, 23]. Next to Leucaena leucocephala, G. sepium may be the most cultivated multipurpose tree [22, 23] with G. sepium often affording the same amount or even more biomass as L. leucocephala [23]. A reason for preferring G. sepium over L. leucocephala is resistance to the defoliating psyllid Heteropsylla cubana [23]. G. sepium tolerates a wide variety of soils, including slightly saline and clay, and resprouts quickly after a fire [22]. On the other hand, G. sepium does not tolerate cooler temperatures well [22, 24] so that it is not found at altitudes exceeding 1200–1600 m with the onset of flowering being influenced by altitude. G. sepium has been reported to have high nutritional value as a protein supplement for animals such as cattle, sheep, and goats [25]. Crude extracts may have antifungal properties and, otherwise, G. sepium has been used for medicinal purposes including treatment of bruises, burns, colds, coughs, fever, ulcers, and other medical issues while, on the other hand, leaves, seeds and powdered bark have been reported to exhibit toxicity to humans under certain conditions [26]. These mixed properties are also demonstrated by cattle, for example, will not necessarily readily devouring the leaves due to emissions of volatiles [24] and that the leaves or ground bark, mixed with corn, are used as a rodenticide [25]. Otherwise, G. sepium finds significant use as a source of wood for fuel, shade for plantation crops, green manure, living fences, construction poles, fodder, bee forage and other purposes.

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J Am Oil Chem Soc

G. sepium produces an abundance of seeds. The seeds are shed from the pods by dehiscence up to 25 m [26]. Depending on origin, 75–180 kg/ha of seed can be produced with seven seeds per pod and approximately 8000 seeds/kg [25]. Otherwise, no reliable figures on the production of G. sepium and its seeds appear to be available and although the figures above may imply limited yield, using the seeds in applications such as biodiesel may aid overall economics. While the fatty acid profile of G. sepium seed oil has been reported in two papers [27, 28], no reports on its use as a potential feedstock for biodiesel appear to exist nor do reports on other uses of the seed oil. To the best of our knowledge, no comprehensive report exists yet on the methyl esters of G. sepium seed oil for fuel use. Thus, this work constitutes the first report on the properties of G. sepium methyl esters (GSME) as biodiesel fuel. The fatty acid profile is also reported and compared to previous literature. Such data are essential for a database on biodiesel fuels and assessing the most suitable sources and fuel composition.

Experimental Seeds of G. sepium were collected in the vicinity of Sitio Loma, Biñan, Laguna, Philippines (approximate geographical coordinates 14°16′N 121°05′E). Green and brown seeds were collected from February to April 2014, which is the flowering and fruiting season of G. sepium. The oil was extracted from the seeds with hexane in a Soxhlet extractor giving an oil content of 11.3 % (green seeds) and 13.8 % (brown seeds). The fatty acid profile of G. sepium oil as given in Table 1 was determined by preparing the methyl esters using gas chromatography and also picolinyl esters by gas chromatography–mass spectrometry as described previously [29]. The equipment was a Perkin-Elmer (Norwalk, CT, USA) Clarus 580 gas chromatograph (GC) with flame ionization detection utilizing a DB-88 [(88 % cyanopropyl) methylarylpolysiloxane] column (30 m × 0.25 mm ID × 0.20 lm film thickness). The initial temperature of 150 °C was held for 15 min, increased to 210 °C at 2 °C/min, then 50 °C/ min to 220 °C held for 5 min with He as the carrier gas at 9.6 mL/min. The injector and detector temperatures were 240 and 280 °C, respectively. For mass spectrometric analysis by electron ionization, the same type of column and temperature program were applied in an Agilent (Santa Clara, CA, USA) 6890 GC equipped with an Agilent 5973 mass selective detector. The green and brown seeds did not display significant differences in their fatty acid profiles and the oils were therefore combined. Nuclear magnetic resonance (NMR) spectra were obtained with CDCl3 as solvent on a Bruker (Billerica, MA, USA) Avance 500

J Am Oil Chem Soc Table 1  Fatty acid profile of Gliricidia sepium oil investigated here and comparison to prior literature Fatty acid

Present work

[27]

[28]

14:0 16:0 16:1 Δ9 16:2n-4

0.1 16.2 0.1 –

– 15.13 0.37 1.97

– 16.00 ± 0.30 0.2 ± 0.20 –

17:0 18:0 18:1 Δ9 18:1 Δ11 18:2 Δ9,Δ12 18:3 Δ9,Δ12,Δ15 18:3 Δ6,Δ9,Δ12 20:0 20:1 Δ11 22:0 24:0

0.2 14.5 13.3 1.0 48.9 1.4 – 2.3 – 1.0 0.2

– 16.21 23.98 0.66 28.47 1.43 0.31 3.20 0.22 1.65 1.27

– 19.20 ± 0.10 23.80 ± 0.50 – 32.30 ± 0.30 1.20 ± 0.30a – 3.50 ± 0.60 0.20 ± 0.20 1.80 ± 0.20 1.90 ± 0.20

Other

0.8

5.13

a   Given as C18:3 and assumed to be α-linolenic acid for the purposes of the present work

spectrometer operating at 500 MHz (1H-NMR) or 125 MHz (13C-NMR). The oil as extracted had an acid value of 4.4 mg KOH/g, necessitating acid pretreatment with H2SO4 and methanol [30] to prepare methyl esters from the free fatty acids and thereby avoiding issues such as formation of emulsions during the following base-catalyzed transesterification. After this acid pretreatment, the acid value was reduced to 0. Then the Gliricidia sepium oil (60 g scale) was transesterified under standard conditions using 1 % sodium methoxide as catalyst, and a 6:1 molar ratio of methanol to oil at 65 °C for 1 h. After that time, the reaction mixture was allowed to cool to room temperature with phase separation occurring during this time. The phases were separated and the upper methyl ester phase was then washed with water until neutral by slowly dripping the methyl esters into the water and permitting them to rise above the water. This method of washing, repeated as often as necessary to achieve the neutral pH, prevents emulsions which may form when shaking the methyl esters with water. Finally, the methyl esters were dried with magnesium sulfate. Free and total glycerol of the product was analyzed with an Agilent Technologies (Santa Clara, CA, USA) 7890A gas chromatograph (GC) equipped with a cool on-column injector and Agilent Technologies DB-5HT (high-temperature) column as described in the standard method ASTM D6584 [31]. Cetane numbers (CN) were determined at Southwest Research Institute (San Antonio, TX, USA) as derived

CN (DCN) using an Ignition Quality Tester (IQT; ASTM D6890) as described previously [32]. The DCN is an approved alternative to the traditional CN standard (ASTM D613) in the biodiesel standard ASTM D6751. Otherwise, the following methods and equipment were used for pro­ perty determination: (1) ASTM D445 for kinematic visco­ sity with Cannon–Fenske (State College, PA, USA) visco­ meters [33]; (2) EN 14112 [34] for oxidative stability with a Rancimat instrument (Metrohm; Herisau, Switzerland) equipped with software for statistical evaluation; (3) ASTM D6079 for lubricity utilizing a high frequency reciprocating rig (HFRR) lubricity tester [35]; (4) a Phase Technology (Richmond, BC, Canada) cloud, pour and freeze point analyzer for cloud point (CP) and pour point (PP); (5) an Anton Paar (Ashland, VA, USA) DMA 4500M density meter for density; (6) Karl Fischer titration with a Metrohm (Herisau, Switzerland) 831 KF coulometer for water content; (7) AOCS (American Oil Chemists’ Society) method Cd3d 63 for acid value; (8) a Perkin-Elmer (Waltham, MA, USA) Optima 7000 DV ICP-OES spectrometer for analysis of Na, K, P, S, Ca, and Mg.

Results and Discussion Fatty Acid Profile Table  1 gives the fatty acid profile of the seed oil of G. sepium used here as well as the profiles reported previously [27, 28]. The seed oil of G. sepium is characterized mainly by the high content of saturated fatty acids, around 35 %, and high content of linoleic acid around 49 %. No less common or unusual fatty acids such as cyclic, epoxy or hydroxy fatty acids were observed. The absence of such less common fatty acids appears to be typical for most oils from plants of the Fabaceae family but in the seed oils of species of the Albizia and Acacia, which also belong to the Fabaceae family but different genera, fatty acids with cyclic moieties were observed [36]. The fatty acid profile observed in the present work largely agrees with the fatty acid profiles reported previously [27, 28] which also indicate similar amounts of saturated fatty acids (Table 1). The most salient difference to the previously reported fatty acid profiles is the higher content of linoleic acid found here. The content of linoleic acid as discussed in the previous literature was 28.5–32.3 % and compensated for by elevated amounts of oleic acid in the present work (Table 1). Besides hexadecadienoic acid reported previously [27] not being found in the present work, a few other minor differences between the three fatty acid profiles discussed here are obvious in Table 1. The GSME synthesized here were also analyzed by NMR. The following peaks were observed in 1H-NMR,

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J Am Oil Chem Soc

agreeing with the 1H-NMR spectra of other plant oil esters consisting of the same fatty acids: 5.31–5.43 ppm (–CH=CH–), 3.68 ppm (CH3–O–CO–), 2.75–2.80 ppm (–CH=CH–CH2–CH=CH–; t), 2.28–2.34 (CH3–O–CO– CH2–; t), 2.00–2.10 (–CH2–CH2–CH=CH–; m), 1.60–1.68 (CH3–O–CO–CH2–CH2–; m), 1.24–1.40 ((–CH2)x–; m), 0.96–1.0 (–CH=CH–(CH2)–CH3; w) 0.87–0.92 (–(CH2)y– CH3). Integration of these peaks according to a method described in the literature [37] agrees well with these results, giving a total of 34.9 % saturated fatty acids vs 34.5 % as can be calculated for the present data in Table 1. The amounts of individual unsaturated fatty acids also compare well, for example, monounsaturated fatty acids at 15.4 % by NMR vs 14.4 % as given in Table 1 and diunsaturated fatty acids at 47.6 % by NMR vs 48.9 % as given in Table 1. In 13C NMR, characteristic peaks were observed at 174.31 and 174.26 ppm (–O–CO–; several peaks due to the existence of more than one kind of chain), several peaks in the range 130.20–127.91 ppm (–CH=CH–), 51.40 ppm (CH3–O–CO–), 34.11, 34.09 ppm (both O–CO–CH2–), 31.93, 31.90, 31.52 (all three –CH2–CH2–CH3), numerous peaks in the range 29.76–29.08 ppm (all –(CH2)x–), 27.20, 27.18, 27.16 (weak) (all three –CH2–CH=CH–CH2–), 25.63, 24.96, 24.94 (both O–CO–CH2–CH2–), 22.68 and 22.57 ppm (both –CH2–CH2–CH3), 14.10 and 14.05 ppm (both –CH2–CH3). The NMR results thus confirm the data obtained by GC and GC–MS regarding fatty acid profile analysis. Fuel Properties of GSME Cetane Number The CN of a mixture of FAME using the CN of the individual component can be calculated using an equation accounting for proportional contribution of the individual components to the overall CN [38]. This equation is

CNmix =



AC × CNC

(1)

in which CNmix is the CN of the mixture, AC = the relative amount (%) of an individual neat ester in the mixture (as determined by, for example, GC), and CNC the CN of the individual neat ester. According to this equation, the CN of GSME can be calculated as approximately 61. The experimental value was determined (as DCN) to be even higher at 67.5. The high CN of GSME coincides with the high content of saturated FAME which have high CN. The CN of methyl palmitate is approximately 85, the CN of methyl stearate is about 100, and that of methyl eicosanoate has been approximated to be around 115, while the CN of methyl docosanoate and methyl tetracosanoate are even higher [38], accounting for the high CN of GSME.

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Kinematic Viscosity The kinematic viscosity of GSME was determined as 4.38 mm2/s, which is well within the range of kinematic viscosity values prescribed in the biodiesel standards ASTM D6751 and EN 14214 (Table 2). This value is also in excellent agreement with the result of approximately 4.4 mm2/s that could be calculated according to an equation [39] similar to the one used for the CN in which the individual components of the FAME mixture contribute proportionally to their amounts to the overall kinematic viscosity using viscosity data of the individual components [33]. These kinematic viscosity data (40 °C) are 4.38 mm2/s for methyl palmitate, 5.86 mm2/s for methyl stearate, 4.51 mm2/s for methyl oleate, and 3.65 mm2/s for methyl linoleate. For the long-chain saturated FAME, kinematic viscosity contributions to the overall kinematic viscosity can be extrapolated to be 7.40 mm2/s for methyl eicosanoate, 9.31 mm2/s for methyl docosanoate and 11.53 mm2/s for methyl tetracosanoate. Oxidative Stability The oxidative stability of GSME was determined as 2.20 h (Table  2) per the Rancimat test as described in the standard EN 14112. This value is well below the specifications in both biodiesel standards ASTM D6751 and EN 14214. Therefore, GSME would require, as do virtually all biodiesel fuels, the use of antioxidant additives to meet oxidative stability specifications in standards. This is likely a result of the high content of approximately 49 % of methyl linoleate which in the neat form has an oxidative stability of 0.94 h [40]. For the sake of comparison, the relative rates of oxidation of unsaturated FAME given in the literature are 41 for methyl linoleate, 98 for methyl linolenate and even higher for more highly unsaturated fatty esters if the relative rate of methyl oleate is 1 [41]. Furthermore, the oxidative stability of biodiesel (methyl esters) is generally lower compared to the parent vegetable oil which has been explained by the loss of natural antioxidants during the transesterification reaction. Cold Flow The CP of GSME was determined to be 21 and 22.4 °C in two tests with the PP at 19 °C. This is one of the highest CP determined for a biodiesel fuel, even higher than that of palm oil methyl esters, which contain slightly more than 40 % methyl palmitate and methyl stearate combined, determined at around 16 °C [42] and higher than that of the methyl esters of Moringa oleifera which exhibited a CP of 18 °C with 6.5 % methyl palmitate, 6 % methyl stearate 4 % methyl eicosanoate, and 7.1 % methyl docosanoate [9]. Besides the content of methyl

J Am Oil Chem Soc Table 2  Properties of Gliricidia sepium methyl esters with comparison to biodiesel standards Property

G. sepium methyl esters

ASTM D6751

EN 14214

Cetane number Kinematic viscosity (mm2/s) Oxidative stability (h) Cloud point (°C) Pour point (°C) Acid value (mg KOH/g) Lubricity (HFRR wear scar; μm) Density 15 °C (kg/m3) Free glycerol (mass %) Total glycerol (mass %) Sodium (mg/kg) Potassium (mg/kg) Phosphorus (mg/kg) Sulfur (mg/kg) Magnesium (mg/kg)

67.5a 4.38 2.20 21.0, 22.4f 19, 19f 0 140 879.5 0.005, 0.002f 0.028, 0.023f 0.154 0.189 0 5.175 0

47 min 1.9–6.0 3 min –b –b 0.5 max 520 maxc – 0.02 max 0.24 max

51 min 3.5–5.0 8 min –b –b 0.5 max 460 maxd 860–900 kg/m3 0.02 max 0.25 max

Na + K 5 ppm (μg/g) max combined

Na + K 5 mg/kg max combined

0.001 % mass max 0.0015 % mass (ppm) maxe

4 mg/kg max 10 mg/kg max

Calcium (mg/kg)

0

Mg + Ca 5 ppm (μg/g) max combined

Mg + Ca 5 mg/kg max combined

min minimum, max maximum a

  Calculated value is 61. See text

b

  Report of cloud point in ASTM D6751, no limits prescribed. Cold-filter plugging point with varying limits in EN 14214

c

  Per ASTM D975

d

  Per EN 590

e

  For blending with 15 ppm ULSD. 0.05 mass % for blending with 500 ppm sulfur diesel fuel

f

  Duplicate determination

palmitate and methyl stearate in GSME (combined 35 %), this result must largely be traced to the content (approximately 4 %; see Table 1) of even higher melting methyl eicosanoate (m.p. = 46.4 °C [43]), methyl docosanoate (m.p. = 53.2 °C) and methyl tetracosanoate (m.p. = 58.6 °C). This observation agrees with previous work showing that the CP of a mixture methyl esters depends mainly on the nature and amounts of the saturated fatty esters [44] but also shows that minor to moderate amounts of FAME with ≥C20 cause especially high CP. For comparison purposes, soy methyl esters with approximately 10–11 % C16:0 and 4–5 % C18:0 have a CP around 0 °C [45]. Thus, the low-temperature properties of GSME are likely its greatest technical problem. Density The density at 15 °C of GSME was determined as 0.8795 g/ cm3. The density value of GSME is therefore well within the range of the specification in the biodiesel standard EN 14214 with density not being included in ASTM D6751. The value also agrees well with the calculated density (at 15 °C) of approximately 0.8756 according to an equation [46] analogous to those for calculating CN and kinematic viscosity as discussed above.

Lubricity Agreeing with the well-known excellent lubricity of neat biodiesel [47, 48], GSME displays excellent lubricity as well. The wear scar in the HFRR lubricity test [35] was 140  μm (Table 2), well below the maximum values prescribed in the standards ASTM D975 [49] and EN 590 [50] for petrodiesel used as surrogates as biodiesel standards do not contain lubricity specifications. Other Specifications Besides the analyses conducted above, the levels of the six heteroelements Na, K, S, P, Ca, and Mg limited in biodiesel standards were also analyzed (Table 2). None of the limits specified for these elements in biodiesel standards were exceeded. The acid value was determined as 0, thus also meeting specifications in biodiesel standards.

Summary and Conclusions After acid pre-treatment of the parent oil, conventional base-catalyzed transesterification using sodium methoxide

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as catalyst yielded the methyl esters of Gliricidia sepium oil. While GSME meets specifications in biodiesel standards, low-temperature properties are problematic due to high content of saturated FAME. Acknowledgments  The authors thank Kevin R. Steidley for excellent technical assistance, Kim Ascherl for ICP analysis, and Dr. Karl Vermillion for obtaining the NMR spectra, all of USDA/ARS/ NCAUR, and Dr. Michael S. Wibbens (Southwest Research Institute, San Antonio, TX) for cetane number determination.

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