Production of Biodiesel and Bioethanol from the

Journal of Biofuels DOI : 10.5958/0976-4763.2014.00010.5

Vol. 5 Issue 2, July-December 2014 pp. 76-82

Production of Biodiesel and Bioethanol from the Legumes of Leucaena leucocephala Abdul Majeed Khan*, Sher Ali Research Laboratory of Bioenergy and Medicinal Chemistry (RLB-MC), Department of Chemistry, Federal Urdu University, Gulshan-e-Iqbal Campus, University Road, Karachi-75300, Pakistan *Corresponding author e-mail id: [email protected]

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ABSTRACT This research article is based on the production of biodiesel and bioethanol from Leucaena leucocephala. The legumes of L. leucocephala were collected, dried, grinded and soaked in non-polar solvent (n-hexane) and then after a week it was decanted as n-hexane extract, which was evaporated by the rotary evaporator. It was passed through the column chromatography for its purification. The purified oily content was subjected to transesterification by different methods, which yielded fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE) with different percentage yields. All the reactions were conducted under completely anhydrous condition with fast stirring on hot plate. The biodiesels were identified in comparison with American Society for Testing Materials (ASTM). Similarly, for the production of bioethanol, the residue of the plant was soaked in cold water, hot water, 20% H2SO4 and 20% NaOH solution. All of the extracts were evaporated under sunlight. The extracts were subjected to fermentation using the yeast. The bioethanol so produced was identified by the preparation of ethyl benzoate as UV active derivative. Keywords: Leucaena leucocephala, Oil, Transesterification, Biodiesel, Carbohydrates, Fermentation, Bioethanol

1. INTRODUCTION Global warming is much hazardous for living organisms which attracted the researches to develop alternative strategies that can reduce the temperature of the earth. In addition, CO2, CO and some other toxic gases resulted during the burning of fossil fuels and enter into the atmosphere that has the capability to capture the radiations emitted from the sun which causes global warming. Owing to this threat the adverse environmental variations occur. If such risky environmental variations will continue, the green environment will start to wipe out, i.e. no life of animals and plants will experience any safety. In contrast to avoid such drastic situations the production of alternative, renewable, green and environmental friendly biofuels, i.e. biodiesel and bioethanol, can play a vital role. The responsible source that produces different sorts of gases that makes the environmental pollution are vehicles, industries, etc., that utilises fossil fuels in different shapes such as CNG, LPG, diesel, gasoline, etc., as the energy source. The green synthesis has a prominent impact in the reduction of global pollution and warming. Multiple attempts were made to synthesize less causative, inexpensive, qualitative and renewable biofuels from different sources such as algae, chicken fats, sugar as well as different plants such as Eucalyptus and L. Leucocephala[1]. Atmosphere alteration is bringing about by the rising atmospheric content of a series of gases, i.e., CO2, N2O, CH4, O3, and CFCs. The relevant greenhouse gases (GHGs) are all growing as a consequence of human activities so the usage of the fossil energy (i.e. gasoline and diesel) in the transport sector resulted in the discharge of GHGs such as CO2 and CO, which potentially cause climate transform[2, 3]. The responsible factor for the environmental pollution and global warming that produces the CO2 in a very large amount is the burning of fossil fuel[4]. The temperature of the global surface increases with 0.5°C since 1975[5, 6]. The production of renewable fuels at national and international levels has been enhanced due to increasing fossil fuel demands and environmental un-sustainability[7]. The global warming variations attracted the scientists to develop the alternate and green resources[8, 9]. The biomass utilisation for the production of biofuels is also important to overcome the energy crisis [10]. L. leucocephala (Ipil Ipil) is a fast-growing tree belonging to the family leguminacae, which is cultivated in the tropical region including Pakistan[11, 12]. It has blackish brown hard, brittle coated seeds, and the tree plays an IndianJournals.com

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Production of Biodiesel and Bioethanol from the Legumes of Leucaena leucocephala

important role in multiple uses such as firewood, timber, hay for cattle etc.[13–15]. The seeds of L. leucocephala contain carbohydrates, crude protein and lipids as well as tannin and oxalic acid[16,17]. 2. MATERIAL AND METHODS 2.1. Collection and Pretreatment




The legumes (17 kg) of L. leucocephala were collected from the garden of the Department of Chemistry, Federal Urdu University. The plant was identified by Dr. Muhammad Abid, Associate Professor, Department of Botany, FUUAST. The legumes were dried under shadow for 1 month. The net amount of the dry legumes was 9.6 kg. The legumes were grinded by an electrical grinder that produced 7.3 kg powder (Figure 1).

Figure 1: Tree of L. Leucocephala

2.2. Biodiesel 2.2.1. Extraction of fats and oil The powder (7.3 kg) was soaked in the non-polar solvent (n-hexane, 12.5 L) for a week. The extract was separated by decantation and the residue was treated twice with the same solvent. n-Hexane (17.5 L) was used for extraction that resulted in the extraction of 7 L of n-hexane extract. The n-hexane extract was concentrated by the rotary evaporator under reduced pressure. 2.2.2. Column chromatography of biodiesel The crude oil was purified by column chromatography using silica gel (mesh size 100–200) as stationary phase and n-hexane: ethyl acetate (10%, 20% and 50%) as the mobile phase. Finally, the column was run with 100% ethyl acetate and all the fractions were concentrated by the rotary evaporator. 2.3. Preparation of Biodiesel (a) By Microwave oven-assisted synthesis First, sodium alkoxides of methanol and ethanol were prepared by treating each alcohol separately with sodium Journal of Biofuels

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Abdul Majeed Khan, Sher Ali

metal at room temperature that produced white solid sodium alkoxides. Now, the alkoxide (10 ml each) and fat/ oil (2 g) were taken in a quick fit round bottomed flask (100 ml capacity) and then placed in a modified microwave oven (fitted with a reflux condenser) for 5 min. The product mixture was allowed to cool down at room temperature, and then the biodiesel was extracted by the addition of n-hexane (15 ml), chloroform (15 ml) and distilled water (15 ml). The layers were separated by a separating funnel. An organic solvent layer contains biodiesel whereas the water layer contains soap, glycerol and un-reacted alkoxide. The solvent was evaporated by rotary evaporator under reduced pressure that yielded 0.29 g of fatty acid methyl ester (FAME) and 0.09 g of fatty acid ethyl ester (FAEE). During transesterification with methanol and ethanol 0.61 g and 0.92 g soap was obtained respectively. (b) By conventional heating Biodiesel was also prepared by acidic and basic hydrolysis. Fat/oil (2 g) and HCl conc. (5 drops) or NaOH (0.2 g powder) were taken separately in the Erlenmeyer flask containing alcohol (10 ml) and fitted with the condenser. Now, the reaction mixtures were refluxed on hot plate under fast stirring for 12 h. After the reaction, the product mixtures were allowed to cool down on room temperature. The biodiesel so formed was extracted with n-hexane and concentrated by the rotary evaporator.




2.4. Bioethanol For the extraction of carbohydrates, the residue was soaked in cold water (12 L), hot water (4 L), 20% NaOH (5 L) and 20% H2SO4 (5 L). All the solutions were heated on water bath for 1 h except cold water. The solutions were allowed to cool at room temperature and the extracts were decanted. The extracts were evaporated under sunlight for 25–30 days. The pH of each extract was determined and maintained at 7. The carbohydrates were detected by the Benedict test. Each concentrated extract (3 L) was subjected to the fermentation under the surface of the earth using yeast (250 g) for 2 weeks. After fermentation, 300 ml of each sample was separately treated with benzoic acid (2 g) using HCl (0.5 ml) as the catalyst. All the solutions were refluxed on water bath for 12 h. Now, the product mixtures were allowed to cool down on room temperature. Ethyl benzoate was extracted by adding nhexane (50 ml) and distilled water (50 ml). Two layers were separated by a separating funnel. The n-hexane layer containing ethyl benzoate was concentrated on water bath that yielded, ethyl benzoate for cold water (0.89 g), hot water (0.83 g), 20% NaOH (0.96 g) and 20% H2SO4 (0.87 g). Ethyl benzoate so formed was identified by TLC examination in comparison with the standard ethyl benzoate using n-hexane: chloroform (9 : 1) as the mobile phase (Figure 2). 3. RESULTS AND DISCUSSION Presently, a number of global challenges including global warming, energy crisis, pollution, biodiversity and global economy are based on the energy technologies. The extensive consumption of non-renewable fuels is the major cause of these threats. The development of the alternative energy sources is the entire need of the global society to overcome these threats. In this regard, biofuel production from the legumes of L. leucocephala can play a vital role. 3.1. Biodiesel (a) Biodiesel synthesis through microwave irradiations The oily contents obtained from the legumes of L. leucocephala were converted to the biodiesel by microwave irradiations, which is the faster technique for the biodiesel production. In this method, methanol or ethanol were treated with sodium metal to form the corresponding alkoxides. In the next step the alkoxides were treated with triglycerides to produce biodiesel as desired product whereas glycerol as the by-product (Figure 3). The yields of biodiesels namely fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE) were found to be 88 % and 78 % respectively (Table 1). 78

Vol. 5 Issue 2, July-December 2014

Production of Biodiesel and Bioethanol from the Legumes of Leucaena leucocephala

Collection of legumes of Leucaena leucocephala Drying, crushing and soaking

Liquid phase (n-hexane extract: 7 Liters)

Solid phase

Evaporation of solvent by rotary evaporator

Soaking in cold water (11 Liters)

Concentrated oily extract

Decantation

Column chromatography Residue

Water extract




Refined oily contents Hot water

Transesterification

Hot water extract

Hydrolysis

Residue

20% H2SO4

Acid hydrolyzed extract

Residue

20% NaOH

Base hydrolyzed extract

Residue

Discarded

Discarded

Microwave assisted synthesis Biodiesel

Concentrated under sun light Bioethanol

Fermentation

Figure 2: General pathway for the production of biodiesel and bioethanol from L. Leucocephala

OCOR ROH, Na OCOR

OH OH +

Microwave irradiation OCOR Triglyceride

OH

3RCOOR + Soap Biodiesel

Glycerol

Figure 3: Chemical conversion of triglyceride to biodiesel through microwave irradiations

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(b) Biodiesel production through conventional heating The oils were also converted to biodiesel by acidic and basic hydrolysis. The acidic hydrolysis was found to be reversible and slow whereas basic hydrolysis was found to be fast and irreversible. The acidic hydrolysis produced biodiesel and glycerol whereas basic hydrolysis produced biodiesel, glycerol and soap (Figure 4). The percentage yields of FAME produced by acidic and basic hydrolysis were found to be 54% and 68%, respectively, whereas FAEE produced by acidic and basic hydrolysis was found to be 48 and 58%, respectively (Table 1). OCOR OCOR

OH ROH, HCl OH

Heat OCOR Triglyceride

+

OH

3RCOOR Biodiesel

Glycerol

OCOR

OH




ROH, NaOH OCOR

OH

OCOR Triglyceride

OH

+

3RCOOR

+

Soap

Biodiesel

Glycerol

Figure 4: Chemical conversion of triglyceride to biodiesel through conventional heating

3.2. Confirmation of Biodiesel The biodiesel (FAME and FAEE) production was confirmed by TLC examination using n-hexane : chloroform : toluene (7:2:1) as mobile phase. The Rf values of FAME and FAEE were found to be 0.28 and 0.24 (after two runs in the same solvent system) respectively (Figure 5). The confirmation was further supported by the determination of parameters according to ASTM standards for biodiesel which were found to be in agreement with the standard values.

Figure 5: FAME (B) and FAEE (C) confirmation by TLC examination in comparison with oil (A)

A

80

B

C


Production of Biodiesel and Bioethanol from the Legumes of Leucaena leucocephala Table 1: Comparison of % yields of biodiesel S.No.

Methods

Biodiesel

Yield (%)

1.

Microwave-assisted synthesis

2.

Conventional heating

Acidic

FAME FAEE FAME

88 78 54

Basic

FAEE FAME

48 68

FAEE

58

3.3. Bioethanol




The carbohydrates and some other polar contents in the legumes of L. leucocephala were separated by using polar solvents such as cold water, hot water, 20% H2SO4 and 20% NaOH. The extracts of cold water, hot water, 20% H2SO4 and that of 20% NaOH were decanted, purified and evaporated under sunlight. The Benedict test was used for the identification of carbohydrates present in different extracts of L. leucocephala. This test was found to be positive for all the extracts. However, 20% NaOH extract showed a more prominent test, which evidenced that the basic hydrolysis is better than other methods of hydrolysis. The pH of all extracts was determined and maintained at 6.5, which is the best pH for the growth of Saccharomyces cerivisiae. To convert the carbohydrates to bioethanol, the anaerobic fermentation was carried out under the surface of the earth using fungi (S. cerivisiae). The alcohol produced by the fermentation was directly converted to its UV-active derivative namely ethyl benzoate. It was prepared by treating the fermented mixture with the benzoic acid under the acidic condition (Fig. 6). The % yields of bioethanol from different extracts such as cold water, hot water, 20% H2SO4 and 20% NaOH extracts were determined, which were found to be 0.14 %, 1.56 %, 1.32 % and 1.45%, respectively (Table 2). Anaerobic fermentation Carbohydrates

Bioethanol

+

CO 2

+

H 2O

Saccharomyces cerivisiae COOCH 2CH3

COOH HCl +

CH 3CH2OH Reflux, 6hours

Benzoic acid

Alcohol

Ethyl benzoate

Figure 6: Confirmation of bioethanol by UV active derivative formation Table 2: Comparison of % yields of bioethanol from different extracts S.No. 1. 2. 3. 4.

Samples Cold water extract Hot water extract 20 % H2SO4 extract 20 % NaOH extract

Yield of bioethanol (%) 0.14 1.56 1.32 1.45

4. CONCLUSION Currently, the consumption of non-renewable energy sources mainly the fossil fuels are greater than all other nonrenewable and renewable sources. Their extensive use is damaging for our environment. They emit a large amount of CO2, which is a green house gas and contribute for global warming. Globally, we are also facing pollution, Journal of Biofuels

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energy crisis, biodiversity and instability of global economy. These are the challenging threats that need urgent attention from the researchers of different fields. In this regard, the research presented in this article can play a vital role. This article is based on the production of biodiesel and bioethanol from L. leucocephala using different methods. The biodiesel was produced from the oily content by two methods namely microwave oven-assisted synthesis and conventional heating. In addition, for the production of bioethanol, carbohydrates were hydrolysed by different methods to simple sugars that were finally used for the production of bioethanol by anaerobic fermentation. ACKNOWLEDGEMENTS The authors are greatly thankful to the administration of Federal Urdu University for the provision of research grant under “Third Mini Research Project”. The Principal author Dr. Abdul Majeed Khan also thankful to Dr. Muhammad Abid, Department of Botany, FUUAST for the identification of plant.




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