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Influence of acyl acceptor blends on the ester yield and fuel properties of biodiesel generated by whole-cell catalysis of cottonseed oil
Fuel 259 (2020) 116258
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Full Length Article
Influence of acyl acceptor blends on the ester yield and fuel properties of biodiesel generated by whole-cell catalysis of cottonseed oil
T
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Preeti Naina, , Sumit K. Jaiswalb, N. Tejo Prakashc, Ranjana Prakashd, Sanjay Kumar Guptaa a
Environmental Engineering, Department of Civil Engineering, Indian Institute of Technology, Delhi, India Department of Environmental Science and Engineering, Marwadi University Rajkot, India c School of Energy and Environment, Thapar Institute of Engineering and Technology, Patiala, India d School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology, Patiala, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biofuel Ester yields Biodiesel Catalysis Cottonseed oil Fatty acids
One of the promising alternatives to fossil fuels, biodiesel, is a renewable fuel which can be defined as methyl or ethyl ester of fatty acid made prominently from vegetable oils or animal fat. The present study outlines the observations on the fatty acid alkyl ester generation by transesterification of cottonseed oil using Aspergillus species as whole-cell biocatalyst. Current work also represents the difference in ester yield on the addition of alcohol blends with two different molar ratios (1:4 or 1:6). Further, the fuel properties viz., density, viscosity, gross calorific value, flash point, cloud point and pour point of ethyl-butyl and ethyl-propyl ester were analyzed, as per ASTM 6751. No significant difference was observed in ester yield with both the molar ratio of alcohols. Ester yield up to 91% and 87% was obtained using 1:4 and 1:6 M ratio respectively. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Alcohol other than ethanol facilitated only a marginal increase in ester yield with no significant variation from propanol to octanol. The fuel properties showed significant improvement in comparison to standard biodiesel. The use of whole cell bio-catalysis, as demonstrated in present study can be considered as a promising alternative to a chemical catalyst for the generation of long-chain fatty acid esters.
1. Introduction A high demand for energy consumption, along with the environmental issues concerned with the use of fossil fuels has encouraged the development of biodiesel as an alternative source of energy [1]. Biodiesel is a mixture of different fatty acid alkyl esters obtained by the process of transesterification of vegetable oils or animal fats with alcohols [2]. Biodiesel is eco-friendly, non-toxic, free from sulfur and can be sourced from renewable sources. Also, it has a high flash point which is helpful during transportation and storage. Recently, this alternative biofuel has been widely used as an additive in fossil-based diesel in various proportions. However, problems related to biodiesel are high in NOx emissions and engine deposits that continue to gain attention [3] along with paramount interest towards quality and fuel properties of biodiesel for its successful commercialization. Studies conducted by various research groups around the world confirmed that blends of biodiesel with petro-diesel up to a certain limit can be used in diesel engines without any modifications [4]. Biodiesel with appropriate blending also shows significant reduction in emission
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of unburned hydrocarbons, carbon monoxide and particulates [5]. Alcohols used in transesterification have shown notable effects on physicochemical properties of biodiesel and use of higher chain alcohols as acyl acceptor can enhance the biodiesel fuel properties. Biodiesel can be produced both by chemical and biological approach [6]. Chemical approach includes the use of either acid or alkali catalyst whereas biological approaches involves the use of pure or crude lipase enzymes or whole cells. At the commercial level, alkali catalysis is used in the transesterification process and the extent of ester yield is determined by the alcohol used. Hossain et al., [7] reported that in the presence of alkali catalyst, the extent of transesterification decreases with an increase in chain length of alcohols. The emulsion formed during methanolysis breaks down easily to a higher density glycerol layer and lower density methyl ester layer. However, during ethanolysis and butanolysis, the emulsions formed are more stable due to the presence of larger non-polar groups making the separation and purification of biodiesel more difficult [8]. In contrast, during biocatalysis, ester yield increases with increase in chain length of alcohols [9] due to the presence of water that facilitates the catalytic activity of
Corresponding author at: Environmental Engineering Laboratory, Department of Civil Engineering, Indian Institute of Technology, Delhi, India. E-mail address: [email protected] (P. Nain).
https://doi.org/10.1016/j.fuel.2019.116258 Received 14 June 2019; Received in revised form 11 September 2019; Accepted 21 September 2019 0016-2361/ © 2019 Elsevier Ltd. All rights reserved.
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6 h. Blend of ethanol with other alcohols (methanol to octanol) were prepared by mixing in equimolar ratio and subsequent to the hydrolysis reaction, these alcohol blends were added separately as acyl acceptor in the molar ratio of 1:4 and 1:6 (oil: alcohol). In order to avoid the inactivation of lipase enzyme present in fungal biomass, the alcohol blends were added in three parts at regular interval of 12 h. On completion of alcohol addition, followed by 12 h additional incubation. The total time of hydrolysis and esterification was 156 h. All the transesterification reactions were carried out in duplicate. Progress of the alkyl ester formation was monitored at regular intervals by TLC followed by quantification by 1H NMR.
lipases and also maintains their structural integrity [10]. With reference to the application of biodiesel at user-end, one of the foremost concerns is its unfavorable cold flow properties. In cold climates, it is a challenge for automobiles running with biodiesel as this tends to gel (freeze) at lower temperatures, which make un-suitable for use in the regions with low ambient temperatures. The actual temperature at which biodiesel freezes depends on the type of oil or fat and acyl acceptor from which it is made. Unlike petroleum diesel, flow properties of fatty acid methyl ester and ethyl ester (FAME /FAEE) at low temperatures, inhibit their use in cold climatic conditions. A diesel fuel’s cold-weather characteristics are measured by the cloud point (CP) and the pour point (PP) value. Fossil fuels have a broader range of temperatures (≈20 °C) among the CP and the PP. However, when compared to FAME/FAEE, fatty acid butyl esters show improved cold flow properties [5]. Ester of long-chain alcohols also possesses higher carbon to hydrogen ratio compared to FAME/FAEE that positively influences the calorific value of biodiesel. Majority of the studies carried out on biocatalyzed transesterification, reported till-date, have been carried out using methanol to butanol as acyl acceptor [5]. However, biocatalysed tranesterification is also a preferred preferred approach, over alkali catalyst transesterification, while using higher chain alcohols as acyl acceptors. To the best of our knowledge, there are no reports available on the application of biocatalyst for generation of alkyl ester using higher alcohols (beyond butanol) and characterization of their blends. Microorganisms such as Aspergillus sp. and Rhizopus oryzae have been used to demonstrate the application whole-cell bio-catalysts as alternative to cost-intensive nature associated with application of pure enzymes as biocatalysts, to carry out transesterification reaction [11–15]. Microorganisms act as biocatalysts by transforming hydrocarbon chains into to lipids metabolically and stored as triacylglycerols and esters. Further, the application of microorganism in biodiesel production is relatively cheap and easy-to-scale up approach in long term. The aim of the present study was 1) to evaluate the potential of isolated fungus Aspergillus sp., reported in our earlier studies, as wholecell biocatalyst to carry out transesterification of cottonseed oil; 2) to investigate the effect of mixture of ethanol and other alcohols (methanol to octanol) as acyl acceptors; 3) to investigation the effect of two different ratios of oil to alcohol viz., 1:4 and 1:6, on percentage yield of total ester (ethyl ester, and corresponding alkyl esters) and 4) to analyses the fuel properties such as cloud point, pour point and viscosity of alkyl esters with maximum yield.
2.3. Analysis The hydrolytic reaction and ester formation at various intervals were constantly monitored by TLC for the conversion of oil to free fatty acids until the oil disappeared completely. Silica gel was used as stationary phase and hexane: ethyl acetate (9:1) as a mobile phase [18]. The chromatogram was developed in the iodine chamber. After completion of transesterification reaction, alkyl esters were separated through filtration and remaining alcohol was evaporated using rotary evaporator. Different alkyl esters present in the samples were quantified using 1H NMR (400 MHz; Jeol JNM-ECS 400). CDCl3 (deuterated chloroform) was used as solvent and tetramethyl silane as an internal standard. 1H NMR spectra were recorded with a pulse duration of 2.18 s with a relaxation delay of 4 s and 16 scans. 2.4. Determination of fuel properties of fatty acid esters relevant to biodiesel Compared to other alcohol blends, higher ester yield was obtained in the case of ethyl-propyl and ethyl-butyl. Therefore, these two samples were further subjected to the study of fuel properties. Fuel properties, namely calorific value (IS:1350 (P-II), 1970) flashpoint (IS:1448 (P-21), 1992), pour point (IS:1448 (P-10), 1970), kinematic viscosity (IS:1448 (P-25), 1976), ash content (IS:1448 (P-4)), density (IS:1448 (P16), 1990), FFA as oleic acid (SP:18 (P-13), 1984), sulphur content (IS:1448 (P-33), 1991), sediment (IS:1448 (P-30), 1970) and cloud point (IS:1448 (P-10), 1970) were carried out. 3. Results and discussion The present study was aimed at optimizing the molar ratio (1:4 and 1:6) of different blends of alcohols with ethanol as a common component of the blend so as to obtain enhanced transesterification and improved fuel properties using whole-cell catalyst.
2. Experimental methodology 2.1. Fungal biocatalyst
3.1. Quantification of different alkyl esters by 1H NMR
Aspergillus sp. was isolated by our group from contaminated butter and was reported to exhibit significant oil tolerance and transesterification potential [16,17]. For the generation of fungal biomass, the test fungal spores were inoculated aseptically in 500 ml Erlenmeyer flask containing 250 ml of sterile potato dextrose broth (PDB) and incubated at 30 °C, 150 rpm for 72 h. The active fungal biomass obtained from PDB was further used as a biocatalyst to carry out transesterification reaction. Fig. 1 shows the methodology followed in present study in detail.
In the present study, the sample generated after transesterification containing a mixture of fatty acid alkyl esters and individual ester yield was quantified by 1H NMR. In 1H NMR spectrum, the amplitude of a hydrogen peak is proportional to the number of hydrogen nuclei present in the molecule and each peak indicates different environments of hydrogen atoms in a molecule. Fig. 2 shows a typical 1H NMR spectrum of ethyl-butyl ester blend. After alcoholysis, if the sample contains oil, the methylene hydrogen of glycerol (eCH2OCOR) in unreacted oil and the ethoxy hydrogen (eOCOCH2CH3) of ethyl ester are superimposed in the region of 4.09–4.15 ppm as a quadrate. Unlike ethoxy hydrogen (quadrate), the signal of alcoxy hydrogen (eOCOCH2CH2CH2e) of other alkyl esters (propyl onwards) appears at lower δ value (4.05–4.08 ppm) as a triplet (Fig. 2). Similarly, in the case of methylethyl ester blend, the signal of methoxy hydrogen (eOCOCH3) appears at 3.66 ppm as a singlet (Fig. 3). The signal of α-acyl methylenic hydrogen (eOCOCH2e) of all esters and unreacted FFA and oil lie in the range of 2.22–2.24 ppm. For the quantification of ester yield, different
2.2. Transesterification of oil using different alcohol blends In all experiments, approximately 20 g (wet weight) of active fungal biomass was inoculated in 250 ml Erlenmeyer flask containing 30 ml aqueous medium (Bushnell-Haas broth, 0.5% w/v mycological peptone and 0.5% w/v bi-ammonium hydrogen orthophosphate) and 70 ml cottonseed oil as main carbon source. Culture flask was further incubated at 30 °C at 150 rpm for 120 h for the hydrolysis of oil which was monitored using thin-layer chromatography (TLC) at an interval of 2
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Fig. 1. Methodology schematic for present study.
mathematical derivations have been used. Individual percentage of ethyl and other alkyl esters (propyl onwards) in blends were quantified by formula proposed by Silva et al. [19] (Eq. (1)) and Sharma et al. [20] (Eq. (2)) respectively, where integral values of alcoxy hydrogen of individual ester and α-acyl methylenic hydrogen were taken into consideration.
− ITAG ⎞ I %CE = 100 × ⎛⎜ TAG + EE ⎟ IαCH2 ⎝ ⎠
(1)
The notations in this equation are % CE = percentage conversion of oil into ethyl ester; ITAG = integration of glyceryl methylenic hydrogen (eCH2OCOR) of oil at 4.28–4.32 ppm; ITAG+EE = joint integration of glyceryl methylenic hydrogen of oil and ethoxy hydrogen of ethyl ester at 4.09–4.15 ppm (quadrate); and IαCH2 = integration of α-acyl
Fig. 2. 1H NMR spectra of ethyl-butyl ester. 3
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Fig. 3. 1H NMR spectra of methyl-ethyl ester.
methylenic hydrogen (eOCOCH2e) in oil and ethyl esters over the range of 2.2–2.4 ppm (Fig. 2).
(n = 3) across two different blends of various alcohols.
AEα −CH2 ⎞ %CA = 100 × ⎛⎜ ⎟ ⎝ Aα − CH2 ⎠
3.2. Effect of oil to alcohol molar ratio (2)
During transesterification, the stoichiometric ratio of oil to alcohol is 1:3, [22] however, a slight excess of alcohol is required to drive the reaction in forwarding direction [23]. Generally, transesterification reaction mediated by chemical catalyst requires 1:6 to 1:30 oil to alcohol molar ratios [24–26] and it was found that, rate of transesterification decreases with increase in alcohol ratio after a certain limits [27]. Whereas, biological catalysts like lipases or lipase containing cells require lower ratios (up to 1:6) [28–33] to facilitate ester yield more than 90%. In present study, 1:4 oil to alcohol (2 mol ethanol + 2 mol other alcohols) ratio was used to drive the transesterification in forward direction. Similarly, 1:6 M ratio was used to provide 3 mol of ethanol and 3 mol of other alcohols during transesterification to facilitate the availability of individual alcohol in their respective stoichiometric ratio for one mole of oil. The obtained results clearly indicated that, higher chain alcohols were preferred over lower chain during biocatalyzed transesterification even though both alcohols were present in equimolar ratio. Table 1 shows the total and individual alkyl ester yields obtained using 1:4 and 1:6 M ratio. Slightly higher esters yields were observed in the case of 1:4 M ratio as compared to 1:6. Therefore, 1:4 M ratio is
The notations in this equation are %CA = percentage conversion of oil into alkyl ester; AEα-CH2 = integration value of the alcoxy hydrogen of the alkyl esters (triplet, 4.05–4.08 ppm); and Aα-CH2 = integration of α-acyl methylenic hydrogen (eOCOCH2e) in oil and ethyl esters over the range of 2.2–2.4 ppm (Fig. 2). The methyl ester was quantified using the Eq. (3) given by Gelbard et al. [21]
%CM = 100 ×
2IME 3IαCH2
(3)
The notations in this equation are %CM = percentage conversion of oil into corresponding methyl ester; IME = integration value of the methoxy protons of the methyl esters at 3.66 ppm; and IαCH2 = integration value of α-methylene protons over the range of 2.2–2.4 ppm (Fig. 3). Total percentage of ester yield in the samples was calculated by adding a percentage of ethyl ester and their corresponding alkyl ester (methanol to octanol). Table 1 outlines the observations on ester yield Table 1 Percentage of ethyl ester and corresponding ester in different molar ratio. Alcohol in the blend
Total ester (%)
% Ethyl ester
% of Other corresponding ester
Alcohol molar ratio 1:4 Methanol Propanol Butanol Pentanol Hexanol Heptanol Octanol
77.30 91.25 90.28 82.53 84.35 87.30 87.75
1:6 ± ± ± ± ± ± ±
0.5 0.4 1.3 1.4 2.6 1.8 1.7
76.85 87.15 83.60 80.15 74.50 80.70 80.65
1:4 ± ± ± ± ± ± ±
0.8 2.0 1.8 1.3 1.8 2.1 1.5
1:6
46.67 32.70 25.20 32.13 29.05 31.65 24.70
4
± ± ± ± ± ± ±
0.2 0.4 0.2 1.6 2.6 0.5 0.4
45.50 31.45 29.75 27.80 28.80 29.55 27.50
1:4 ± ± ± ± ± ± ±
0.7 2.2 0.5 1.1 0.8 0.6 2.1
30.33 58.50 64.80 50.40 55.30 55.65 63.10
1:6 ± ± ± ± ± ± ±
1.5 0.7 1.4 2.2 1.3 2.3 1.2
31.35 55.85 53.85 52.25 45.70 51.15 53.00
± ± ± ± ± ± ±
1.3 2.3 1.1 1.8 0.8 2.1 1.5
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more favorable to carry out transesterification using whole-cell biocatalyst. Following other studies as well are in accordance with the above justifications. Guldhe et al. [34] has reported synthesis of biodiesel from microalgae (S. obliquus) using immobilized Aspergillus niger whole cell lipase biocatalyst while taking methanol to oil molar ratio of 3:1, 5:1 and 7:1. After optimization, 5:1 M ratio was reported to be optimum amount. In another study, Amoah et al. [35] reported addition of methanol with oil at 1:1 M ratio and further increasing upto a molar ratio of 4:1 (methanol to oil) using Aspergillus oryzae as whole cell catalyst. Almyasheva et al. [36] has also reported stepwise addition of methanol with four molar equivalent (1 M equivalent of methanol in 1st, 2nd, 3rd, & 4th phases) usig Aspergillus niger as whole cell biocatalyst.
Table 2 Different parameters of ethyl-propyl and ethyl-butyl esters produced from cottonseed oil. Parameters
Unit
Ethylpropyl
Ethylbutyl
ASTM D6751 standard for biodiesel
Gross calorific Value (GCV) Flash Point Ash Viscosity @ 40 °C Sediment (%) Sulphur (%)
Kcal/kg
8866
9382
7870
°C (minimum) % w/w centipoise % w/w % w/w (maximum) gm/cc °C °C %
104 ND 6.02 ND ND
71 ND 7.11 ND ND
130 NA 4.0–6.0 0.05 0.05
0.87 −5 −20 91
0.86 −6 −26 90
0.89 NA −15.0 to 10.0 NA
Density @15 °C Cloud point Pour point Ester Content
3.3. Effect of increase in the carbon chain Esters obtained from blends of different alcohols (Table 1), the yield of ethyl ester was lower when compared to their corresponding alkyl ester except for methanol. This clearly indicates that the alcohol with longer carbon chain is preferred over the shorter carbon chain, during the transesterification process, an observation that is notable in the present study. There was a significant (p < 0.05) variation between the yields of ethyl ester over the yield of other alkyl esters in either of the molar ratio (Fig. 4). The present work also supports the previous observations [7,9,33], where unlike chemical catalysts, biocatalysts are more favourable for the synthesis of higher alcohol chain fatty acid esters. Generally, either methanol or ethanol is used as an acyl acceptor during transesterification, but these alcohols are soluble in water and thus inactivate the lipase [37,38]. The solubility of methanol and ethanol in oil is approximately 50% and 75% respectively [39]. The solubility of propanol and other higher alcohols is comparatively higher as they are completely miscible with vegetable oils because of their low polarity compared to methanol and ethanol [40]. As a result, transesterification reactions with alcohols having three or more carbon atoms are monophasic throughout which enhances the rate and extent of the reaction [41,42]. Unlike methanol and ethanol, there are no mass transfer limitations across interfaces between phases in the case of higher alcohols, since all reactants are in a single (oil) phase.
NA: Not available, ND: Not detected.
characteristics of esters obtained from ethanol-propanol and ethanolbutanol were approximately similar as per the standards specified in Table 2. 3.5. Effect on cloud point and pour point Pour point (PP) and cloud point (CP) have been used to estimate the behaviour of diesel fuels in cold weather and lower PP and CP values exhibit improved cold flow properties of biodiesel. In present work, CP and PP of ethyl-propyl and ethyl-butyl ester blends are presented in Table 2. The observed CP and PP of ethyl-butyl are lower than ethylpropyl ester. This indicates that with an increase in the chain length of alcohol portion of fatty acid esters, there is a concomitant decrease in their crystallization temperature. The findings are also supported by the previous report wherein the CP of methyl, ethyl, isopropyl, and 2-butyl esters sourced from soybean oil were −2, −2, −9 and −12 °C, respectively [44] whereas CP of beef tallow sourced methyl, ethyl, propyl, and butyl esters was 17 °C, 15 °C, 12 °C, and 9 °C, respectively [45]. Similarly, blends of higher chain alcohols like butanol, pentanol and hexanol with fatty acid methyl ester generated from waste cooking oil have been shown improved cold flow properties [46]. Other than ester head group, cold flow properties also depend upon the double bond content of oil used for transesterification. Saturated fatty acids exhibit high temperature of crystallization and thus produce biodiesel with inferior cold flow properties. Soybean oil contains 14% saturated fatty acids and beef tallow contains 25% saturated fatty acids. Even though, cottonseed oil contains 26% saturated fatty acids, the CP of ethyl-propyl (−5) and ethyl-butyl ester (−6) was found to be better than soybean oil and beef tallow. Similarly, PP of biodiesel also depends upon the degree of saturation and ester head group [47]. In the current work, the PP values of ethylpropyl and ethyl-butyl esters were −20 °C and −26 °C, which is better, in spite of the presence of 26% saturated fatty acids.
3.4. Effect of blends on fuel properties of ethyl-propyl and ethyl-butyl ester In general practice, alcohol-biodiesel blends are used to overcome the disadvantage of higher viscosity, poor cold flow behaviour for biodiesel and the lower cetane number for alcohols; as the blends observably show better fuel properties compared to biodiesel fuel [43]. After transesterification for a selected ratio of alcohol, a higher ester yield was obtained from ethanol-propanol (91%) and ethanol-butanol (90%) blends. Biodiesel obtained after synthesis were examined for gross calorific value (GCV), flash point, ash, kinematic viscosity, sediment, sulfur content, density, cloud point (CP) and pour point (PP). The
3.6. Effect on flashpoint The flashpoint is the lowest temperature at which the fuel can form an ignitable mixture with air. The flashpoints of ethyl-propyl and ethylbutyl esters were 104 °C and 71 °C, respectively, which are higher than that of diesel fuel, which is 60 °C. This difference is related to the components present in each fuel. Fossil diesel is composed of low molecular weight molecules and a branched compound that leads to the reduction of the flash point. In contrast, among biodiesels the difference in flash point possibly depends upon fatty acid chain length, level of unsaturation, types of alcohol used and residual alcohol present after transesterification. According to ASTM D6751 standard, minimum flash
Fig. 4. Variations in the yields of ethyl ester (EE) and other alkyl esters (other) in both 1:4 and 1:6 M ratios. 5
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well as blends of ethanol-long chain alcohols. Cold flow properties of ethyl-alkyl ester blends were better than properties of standard biodiesel. Alkyl esters with larger ester head groups (such as ethyl, propyl, or butyl) have greater energy content. The viscosity of the ethyl-propyl and ethyl-butyl esters was found to be higher than stearic acid ethyl ester. Also, flash point got improved using alcohol blends compared to standard biodiesel.
point of 100% methyl ester should be 130 °C. In present work, reduction in flash point from ethyl-propyl to ethyl-butyl clearly indicated that alcohol chain length significantly reduce the flash point of biodiesel. A higher flash point indicates that the biodiesel is less flammable than petroleum diesel; hence, biodiesel is safer to handle [48]. 3.7. Effect on viscosity and density
Acknowledgments
Among the biodiesel properties, viscosity and density are the most important parameters that affect the engine performance and the emission characteristics. Higher viscosity affects the fuel atomization during injection into the combustion chamber and increases the carbon deposition on fuel filter, demands more energy from the fuel pump [49]. In the present study, ethyl-propyl and ethyl-butyl esters had viscosities of 6.02 cP and 7.11 cP respectively at 40 °C (Table 2). Factors such as larger ester head group and longer chain length result in high kinematic viscosity. The kinematic viscosity of methyl esters of lauric, myristic, palmitic, and stearic acids is 2.43, 3.30, 4.38, and 5.85 cP respectively and kinematic viscosities of methyl, ethyl, and butyl esters of stearic acid is 5.85, 5.92, and 7.59 cP respectively [5]. In our findings, the viscosity of the ethyl-propyl and ethyl-butyl esters was found to be higher when compared to the stearic acid ethyl ester (5.92). This indicates that a larger head group increases the viscosity of biodiesel. The density of ethyl-propyl and ethyl-butyl ester blends was observed to be 0.87 gm/cc and 0.86 gm/cc which fulfill the prescribed limits. Biodiesel has densities between 0.860 gm/cc and 0.897 gm/cc at 15 °C which is higher than that of petroleum diesel (0.83 gm/cc) [50].
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3.8. Effect on gross calorific value The gross calorific value (GCV) is the total energy released as heat when a fuel undergoes complete combustion with oxygen [51]. In the present study, the GCV of ethyl-propyl and ethyl-butyl ester blends were 8866 and 9382 Kcal/kg respectively which is higher than standard biodiesel (7870 Kcal/kg) (Table 2). The main factor that influences the calorific value or energy content of biodiesel is carbon to oxygen ratio which is directly proportional to chain length [5,49]. Biodiesel fuels with larger ester head groups (such as ethyl, propyl, or butyl) have greater energy content as a result of their greater carbon to oxygen ratio. The present study, thus, demonstrates that higher ester yield can be achieved using a molar ratio of 1:4 or 1:6 (oil: alcohol) and whole-cell fungal system as a biocatalyst for the production of fatty acid alkyl esters. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Biodiesel, thus produced, also showed better cold flow properties with increase in a carbon chain of alcohol. Alcohol other than ethanol facilitates the only marginal increase in ester yield with an increase in the carbon chain. 4. Conclusion The present study investigated production of biodiesel form refined cottonseed oil using Aspergillus sp. as whole-cell biocatalyst, and mixture of ethanol and other alcohols (methanol to octanol) as acyl acceptors. The effect of alcohols blends in two different ratios of oil to alcohol viz., 1:4 and 1:6, on ester yield and its fuel properties was evaluated. The results showed no significant difference in the ester yield with variation in the molar ratio of oil to alcohol from 1:4 to 1:6. Ester yield up to 91% and 87% was obtained using 1:4 and 1:6 M ratio respectively. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Alcohol other than ethanol facilitated only a marginal increase in ester yield with no significant variation from propanol to octanol. Longer chain alcohol was preferred over the shorter chain as evident by increasing yield of ester in the methanol-ethanol blend as 6
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optimized medium. Mycoscience 2018;59:147–52. [37] Kumar D, Das T, Giri BS, Rene ER, Verma B. Biodiesel production from hybrid nonedible oil using bio-support beads immobilized with lipase from Pseudomonas cepacia. Fuel 2019;255:115801. [38] Salis A, Pinna M, Monduzzi M, Solinas V. Biodiesel production from triolein and short chain alcohols through biocatalysis. J Biotechnol 2005;119(3):291–9. [39] Shimada Y, Watanabe Y, Sugihara A, Tominaga Y. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J Mol Catal B: Enzymatic 2002;17(3–5):133–42. [40] Boocock DG, Konar SK, Mao V, Sidi H. Fast one-phase oil-rich processes for the preparation of vegetable oil methyl esters. Biomass Bioenergy 1996;11(1):43–50. [41] Zhou W, Boocock DGB. Phase behavior of the base-catalyzed transesterification of soybean oil. J Am Oil Chem Soc 2006;83(12):1041–5. [42] Zhou W, Boocock DGB. Phase distributions of alcohol, glycerol, and catalyst in the transesterification of soybean oil. J Am Oil Chem Soc 2006;83(12):1047–52. [43] Choi CY, Bower GR. Reitz RD.x Effects of biodiesel blended fuels and multiple injections on DI diesel engines. SAE Trans 2006:388–407. [44] Lee I, Johnson LA, Hammond EG. Use of branched-chain esters to reduce the crystallization temperature of biodiesel. J Am Oil Chem Soc 1995;72(10):1155–60. [45] Foglia TA, Nelson LA, Dunn RO, Marmer WN. Low-temperature properties of alkyl esters of tallow and grease. J Am Oil Chem Soc 1997;74(8):951–5. [46] Atabani AE, Shobana S, Mohammed MN, Uğuz G, Kumar G, Arvindnarayan S, et al. Integrated valorization of waste cooking oil and spent coffee grounds for biodiesel production: blending with higher alcohols, FT–IR, TGA, DSC and NMR characterizations. Fuel 2019;244:419–30. [47] Ali OM, Mamat R, Abdullah NR, Abdullah AA. Effects of blending ethanol with palm oil methyl esters on low temperature flow properties and fuel characteristics. Int J Adv Sci Technol 2013;59:85–96. [48] Laza T, Bereczky Á. Basic fuel properties of rapeseed oil-higher alcohols blends. Fuel 2011;90(2):803–10. [49] Knothe G. Designer biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuel 2008;22(2):1358–64. [50] Bhale PV, Deshpande NV, Thombre SB. Improving the low temperature properties of biodiesel fuel. Renew Energy 2009;34(3):794–800. [51] Demirbaş A. Biodiesel fuels from vegetable oils via catalytic and non-catalytic supercritical alcohol transesterifications and other methods: a survey. Energy Convers Manage 2003;44(13):2093–109.
Fuel 2010;89(1):1–9. [25] Souza GK, Scheufele FB, Pasa TLB, Arroyo PA, Pereira NC. Synthesis of ethyl esters from crude macauba oil (Acrocomia aculeata) for biodiesel production. Fuel 2016;165:360–6. [26] Kostić MD, Bazargan A, Stamenković OS, Veljković VB, McKay G. Optimization and kinetics of sunflower oil methanolysis catalyzed by calcium oxide-based catalyst derived from palm kernel shell biochar. Fuel 2016;163:304–13. [27] Elkelawy M, Alm-Eldin Bastawissi H, Esmaeil KK, Radwan AM, Panchal H, Sadasivuni KK, et al. Experimental studies on the biodiesel production parameters optimization of sunflower and soybean oil mixture and DI engine combustion, performance, and emission analysis fueled with diesel/biodiesel blends. Fuel 2019;255:115791. [28] Dantas JH, De Paris LD, Barão CE, Arroyo PA, Soares CMF, Visentainer JV, et al. Influence of alcohol: oil molar ratio on the production of ethyl esters by enzymatic transesterification of canola oil. Afr J Biotechnol 2013;12(50):6968. [29] Kumari A, Mahapatra P, Garlapati VK, Banerjee R. Enzymatic transesterification of Jatropha oil. Biotechnol Biofuels 2009;2(1):1. [30] Tian X, Dai L, Liu D, Du W. Improved lipase-catalyzed methanolysis for biodiesel production by combining in-situ removal of by-product glycerol. Fuel 2018;232:45–50. [31] Zhang H, Liu T, Zhu Y, Hong L, Li T, Wang X, et al. Lipases immobilized on the modified polyporous magnetic cellulose support as an efficient and recyclable catalyst for biodiesel production from Yellow horn seed oil. Renew Energy 2020;145:1246–54. [32] Marín-Suárez M, Méndez-Mateos D, Guadix A, Guadix EM. Reuse of immobilized lipases in the transesterification of waste fish oil for the production of biodiesel. Renew Energy 2019;140:1–8. [33] Sharma YC, Singh B. Development of biodiesel: current scenario. Renew Sustain Energy Rev 2009;13:1646–51. [34] Guldhe A, Singh P, Kumari S, Rawat I, Permaul K, Bux F. Biodiesel synthesis from microalgae using immobilized Aspergillus niger whole cell lipase biocatalyst. Renew Energy 2016;85:1002–10. [35] Amoah J, Ho S-H, Hama S, Yoshida A, Nakanishi A, Hasunuma T, et al. Converting oils high in phospholipids to biodiesel using immobilized Aspergillus oryzae wholecell biocatalysts expressing Fusarium heterosporum lipase. Biochem Eng J 2016;105:10–5. [36] Almyasheva NR, Shuktueva MI, Petrova DA, Kopitsyn DS, Kotelev MS, Vinokurov VA, et al. Biodiesel fuel production by Aspergillus niger whole-cell biocatalyst in
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Full Length Article
Influence of acyl acceptor blends on the ester yield and fuel properties of biodiesel generated by whole-cell catalysis of cottonseed oil
T
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Preeti Naina, , Sumit K. Jaiswalb, N. Tejo Prakashc, Ranjana Prakashd, Sanjay Kumar Guptaa a
Environmental Engineering, Department of Civil Engineering, Indian Institute of Technology, Delhi, India Department of Environmental Science and Engineering, Marwadi University Rajkot, India c School of Energy and Environment, Thapar Institute of Engineering and Technology, Patiala, India d School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology, Patiala, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Biofuel Ester yields Biodiesel Catalysis Cottonseed oil Fatty acids
One of the promising alternatives to fossil fuels, biodiesel, is a renewable fuel which can be defined as methyl or ethyl ester of fatty acid made prominently from vegetable oils or animal fat. The present study outlines the observations on the fatty acid alkyl ester generation by transesterification of cottonseed oil using Aspergillus species as whole-cell biocatalyst. Current work also represents the difference in ester yield on the addition of alcohol blends with two different molar ratios (1:4 or 1:6). Further, the fuel properties viz., density, viscosity, gross calorific value, flash point, cloud point and pour point of ethyl-butyl and ethyl-propyl ester were analyzed, as per ASTM 6751. No significant difference was observed in ester yield with both the molar ratio of alcohols. Ester yield up to 91% and 87% was obtained using 1:4 and 1:6 M ratio respectively. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Alcohol other than ethanol facilitated only a marginal increase in ester yield with no significant variation from propanol to octanol. The fuel properties showed significant improvement in comparison to standard biodiesel. The use of whole cell bio-catalysis, as demonstrated in present study can be considered as a promising alternative to a chemical catalyst for the generation of long-chain fatty acid esters.
1. Introduction A high demand for energy consumption, along with the environmental issues concerned with the use of fossil fuels has encouraged the development of biodiesel as an alternative source of energy [1]. Biodiesel is a mixture of different fatty acid alkyl esters obtained by the process of transesterification of vegetable oils or animal fats with alcohols [2]. Biodiesel is eco-friendly, non-toxic, free from sulfur and can be sourced from renewable sources. Also, it has a high flash point which is helpful during transportation and storage. Recently, this alternative biofuel has been widely used as an additive in fossil-based diesel in various proportions. However, problems related to biodiesel are high in NOx emissions and engine deposits that continue to gain attention [3] along with paramount interest towards quality and fuel properties of biodiesel for its successful commercialization. Studies conducted by various research groups around the world confirmed that blends of biodiesel with petro-diesel up to a certain limit can be used in diesel engines without any modifications [4]. Biodiesel with appropriate blending also shows significant reduction in emission
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of unburned hydrocarbons, carbon monoxide and particulates [5]. Alcohols used in transesterification have shown notable effects on physicochemical properties of biodiesel and use of higher chain alcohols as acyl acceptor can enhance the biodiesel fuel properties. Biodiesel can be produced both by chemical and biological approach [6]. Chemical approach includes the use of either acid or alkali catalyst whereas biological approaches involves the use of pure or crude lipase enzymes or whole cells. At the commercial level, alkali catalysis is used in the transesterification process and the extent of ester yield is determined by the alcohol used. Hossain et al., [7] reported that in the presence of alkali catalyst, the extent of transesterification decreases with an increase in chain length of alcohols. The emulsion formed during methanolysis breaks down easily to a higher density glycerol layer and lower density methyl ester layer. However, during ethanolysis and butanolysis, the emulsions formed are more stable due to the presence of larger non-polar groups making the separation and purification of biodiesel more difficult [8]. In contrast, during biocatalysis, ester yield increases with increase in chain length of alcohols [9] due to the presence of water that facilitates the catalytic activity of
Corresponding author at: Environmental Engineering Laboratory, Department of Civil Engineering, Indian Institute of Technology, Delhi, India. E-mail address: [email protected] (P. Nain).
https://doi.org/10.1016/j.fuel.2019.116258 Received 14 June 2019; Received in revised form 11 September 2019; Accepted 21 September 2019 0016-2361/ © 2019 Elsevier Ltd. All rights reserved.
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6 h. Blend of ethanol with other alcohols (methanol to octanol) were prepared by mixing in equimolar ratio and subsequent to the hydrolysis reaction, these alcohol blends were added separately as acyl acceptor in the molar ratio of 1:4 and 1:6 (oil: alcohol). In order to avoid the inactivation of lipase enzyme present in fungal biomass, the alcohol blends were added in three parts at regular interval of 12 h. On completion of alcohol addition, followed by 12 h additional incubation. The total time of hydrolysis and esterification was 156 h. All the transesterification reactions were carried out in duplicate. Progress of the alkyl ester formation was monitored at regular intervals by TLC followed by quantification by 1H NMR.
lipases and also maintains their structural integrity [10]. With reference to the application of biodiesel at user-end, one of the foremost concerns is its unfavorable cold flow properties. In cold climates, it is a challenge for automobiles running with biodiesel as this tends to gel (freeze) at lower temperatures, which make un-suitable for use in the regions with low ambient temperatures. The actual temperature at which biodiesel freezes depends on the type of oil or fat and acyl acceptor from which it is made. Unlike petroleum diesel, flow properties of fatty acid methyl ester and ethyl ester (FAME /FAEE) at low temperatures, inhibit their use in cold climatic conditions. A diesel fuel’s cold-weather characteristics are measured by the cloud point (CP) and the pour point (PP) value. Fossil fuels have a broader range of temperatures (≈20 °C) among the CP and the PP. However, when compared to FAME/FAEE, fatty acid butyl esters show improved cold flow properties [5]. Ester of long-chain alcohols also possesses higher carbon to hydrogen ratio compared to FAME/FAEE that positively influences the calorific value of biodiesel. Majority of the studies carried out on biocatalyzed transesterification, reported till-date, have been carried out using methanol to butanol as acyl acceptor [5]. However, biocatalysed tranesterification is also a preferred preferred approach, over alkali catalyst transesterification, while using higher chain alcohols as acyl acceptors. To the best of our knowledge, there are no reports available on the application of biocatalyst for generation of alkyl ester using higher alcohols (beyond butanol) and characterization of their blends. Microorganisms such as Aspergillus sp. and Rhizopus oryzae have been used to demonstrate the application whole-cell bio-catalysts as alternative to cost-intensive nature associated with application of pure enzymes as biocatalysts, to carry out transesterification reaction [11–15]. Microorganisms act as biocatalysts by transforming hydrocarbon chains into to lipids metabolically and stored as triacylglycerols and esters. Further, the application of microorganism in biodiesel production is relatively cheap and easy-to-scale up approach in long term. The aim of the present study was 1) to evaluate the potential of isolated fungus Aspergillus sp., reported in our earlier studies, as wholecell biocatalyst to carry out transesterification of cottonseed oil; 2) to investigate the effect of mixture of ethanol and other alcohols (methanol to octanol) as acyl acceptors; 3) to investigation the effect of two different ratios of oil to alcohol viz., 1:4 and 1:6, on percentage yield of total ester (ethyl ester, and corresponding alkyl esters) and 4) to analyses the fuel properties such as cloud point, pour point and viscosity of alkyl esters with maximum yield.
2.3. Analysis The hydrolytic reaction and ester formation at various intervals were constantly monitored by TLC for the conversion of oil to free fatty acids until the oil disappeared completely. Silica gel was used as stationary phase and hexane: ethyl acetate (9:1) as a mobile phase [18]. The chromatogram was developed in the iodine chamber. After completion of transesterification reaction, alkyl esters were separated through filtration and remaining alcohol was evaporated using rotary evaporator. Different alkyl esters present in the samples were quantified using 1H NMR (400 MHz; Jeol JNM-ECS 400). CDCl3 (deuterated chloroform) was used as solvent and tetramethyl silane as an internal standard. 1H NMR spectra were recorded with a pulse duration of 2.18 s with a relaxation delay of 4 s and 16 scans. 2.4. Determination of fuel properties of fatty acid esters relevant to biodiesel Compared to other alcohol blends, higher ester yield was obtained in the case of ethyl-propyl and ethyl-butyl. Therefore, these two samples were further subjected to the study of fuel properties. Fuel properties, namely calorific value (IS:1350 (P-II), 1970) flashpoint (IS:1448 (P-21), 1992), pour point (IS:1448 (P-10), 1970), kinematic viscosity (IS:1448 (P-25), 1976), ash content (IS:1448 (P-4)), density (IS:1448 (P16), 1990), FFA as oleic acid (SP:18 (P-13), 1984), sulphur content (IS:1448 (P-33), 1991), sediment (IS:1448 (P-30), 1970) and cloud point (IS:1448 (P-10), 1970) were carried out. 3. Results and discussion The present study was aimed at optimizing the molar ratio (1:4 and 1:6) of different blends of alcohols with ethanol as a common component of the blend so as to obtain enhanced transesterification and improved fuel properties using whole-cell catalyst.
2. Experimental methodology 2.1. Fungal biocatalyst
3.1. Quantification of different alkyl esters by 1H NMR
Aspergillus sp. was isolated by our group from contaminated butter and was reported to exhibit significant oil tolerance and transesterification potential [16,17]. For the generation of fungal biomass, the test fungal spores were inoculated aseptically in 500 ml Erlenmeyer flask containing 250 ml of sterile potato dextrose broth (PDB) and incubated at 30 °C, 150 rpm for 72 h. The active fungal biomass obtained from PDB was further used as a biocatalyst to carry out transesterification reaction. Fig. 1 shows the methodology followed in present study in detail.
In the present study, the sample generated after transesterification containing a mixture of fatty acid alkyl esters and individual ester yield was quantified by 1H NMR. In 1H NMR spectrum, the amplitude of a hydrogen peak is proportional to the number of hydrogen nuclei present in the molecule and each peak indicates different environments of hydrogen atoms in a molecule. Fig. 2 shows a typical 1H NMR spectrum of ethyl-butyl ester blend. After alcoholysis, if the sample contains oil, the methylene hydrogen of glycerol (eCH2OCOR) in unreacted oil and the ethoxy hydrogen (eOCOCH2CH3) of ethyl ester are superimposed in the region of 4.09–4.15 ppm as a quadrate. Unlike ethoxy hydrogen (quadrate), the signal of alcoxy hydrogen (eOCOCH2CH2CH2e) of other alkyl esters (propyl onwards) appears at lower δ value (4.05–4.08 ppm) as a triplet (Fig. 2). Similarly, in the case of methylethyl ester blend, the signal of methoxy hydrogen (eOCOCH3) appears at 3.66 ppm as a singlet (Fig. 3). The signal of α-acyl methylenic hydrogen (eOCOCH2e) of all esters and unreacted FFA and oil lie in the range of 2.22–2.24 ppm. For the quantification of ester yield, different
2.2. Transesterification of oil using different alcohol blends In all experiments, approximately 20 g (wet weight) of active fungal biomass was inoculated in 250 ml Erlenmeyer flask containing 30 ml aqueous medium (Bushnell-Haas broth, 0.5% w/v mycological peptone and 0.5% w/v bi-ammonium hydrogen orthophosphate) and 70 ml cottonseed oil as main carbon source. Culture flask was further incubated at 30 °C at 150 rpm for 120 h for the hydrolysis of oil which was monitored using thin-layer chromatography (TLC) at an interval of 2
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Fig. 1. Methodology schematic for present study.
mathematical derivations have been used. Individual percentage of ethyl and other alkyl esters (propyl onwards) in blends were quantified by formula proposed by Silva et al. [19] (Eq. (1)) and Sharma et al. [20] (Eq. (2)) respectively, where integral values of alcoxy hydrogen of individual ester and α-acyl methylenic hydrogen were taken into consideration.
− ITAG ⎞ I %CE = 100 × ⎛⎜ TAG + EE ⎟ IαCH2 ⎝ ⎠
(1)
The notations in this equation are % CE = percentage conversion of oil into ethyl ester; ITAG = integration of glyceryl methylenic hydrogen (eCH2OCOR) of oil at 4.28–4.32 ppm; ITAG+EE = joint integration of glyceryl methylenic hydrogen of oil and ethoxy hydrogen of ethyl ester at 4.09–4.15 ppm (quadrate); and IαCH2 = integration of α-acyl
Fig. 2. 1H NMR spectra of ethyl-butyl ester. 3
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Fig. 3. 1H NMR spectra of methyl-ethyl ester.
methylenic hydrogen (eOCOCH2e) in oil and ethyl esters over the range of 2.2–2.4 ppm (Fig. 2).
(n = 3) across two different blends of various alcohols.
AEα −CH2 ⎞ %CA = 100 × ⎛⎜ ⎟ ⎝ Aα − CH2 ⎠
3.2. Effect of oil to alcohol molar ratio (2)
During transesterification, the stoichiometric ratio of oil to alcohol is 1:3, [22] however, a slight excess of alcohol is required to drive the reaction in forwarding direction [23]. Generally, transesterification reaction mediated by chemical catalyst requires 1:6 to 1:30 oil to alcohol molar ratios [24–26] and it was found that, rate of transesterification decreases with increase in alcohol ratio after a certain limits [27]. Whereas, biological catalysts like lipases or lipase containing cells require lower ratios (up to 1:6) [28–33] to facilitate ester yield more than 90%. In present study, 1:4 oil to alcohol (2 mol ethanol + 2 mol other alcohols) ratio was used to drive the transesterification in forward direction. Similarly, 1:6 M ratio was used to provide 3 mol of ethanol and 3 mol of other alcohols during transesterification to facilitate the availability of individual alcohol in their respective stoichiometric ratio for one mole of oil. The obtained results clearly indicated that, higher chain alcohols were preferred over lower chain during biocatalyzed transesterification even though both alcohols were present in equimolar ratio. Table 1 shows the total and individual alkyl ester yields obtained using 1:4 and 1:6 M ratio. Slightly higher esters yields were observed in the case of 1:4 M ratio as compared to 1:6. Therefore, 1:4 M ratio is
The notations in this equation are %CA = percentage conversion of oil into alkyl ester; AEα-CH2 = integration value of the alcoxy hydrogen of the alkyl esters (triplet, 4.05–4.08 ppm); and Aα-CH2 = integration of α-acyl methylenic hydrogen (eOCOCH2e) in oil and ethyl esters over the range of 2.2–2.4 ppm (Fig. 2). The methyl ester was quantified using the Eq. (3) given by Gelbard et al. [21]
%CM = 100 ×
2IME 3IαCH2
(3)
The notations in this equation are %CM = percentage conversion of oil into corresponding methyl ester; IME = integration value of the methoxy protons of the methyl esters at 3.66 ppm; and IαCH2 = integration value of α-methylene protons over the range of 2.2–2.4 ppm (Fig. 3). Total percentage of ester yield in the samples was calculated by adding a percentage of ethyl ester and their corresponding alkyl ester (methanol to octanol). Table 1 outlines the observations on ester yield Table 1 Percentage of ethyl ester and corresponding ester in different molar ratio. Alcohol in the blend
Total ester (%)
% Ethyl ester
% of Other corresponding ester
Alcohol molar ratio 1:4 Methanol Propanol Butanol Pentanol Hexanol Heptanol Octanol
77.30 91.25 90.28 82.53 84.35 87.30 87.75
1:6 ± ± ± ± ± ± ±
0.5 0.4 1.3 1.4 2.6 1.8 1.7
76.85 87.15 83.60 80.15 74.50 80.70 80.65
1:4 ± ± ± ± ± ± ±
0.8 2.0 1.8 1.3 1.8 2.1 1.5
1:6
46.67 32.70 25.20 32.13 29.05 31.65 24.70
4
± ± ± ± ± ± ±
0.2 0.4 0.2 1.6 2.6 0.5 0.4
45.50 31.45 29.75 27.80 28.80 29.55 27.50
1:4 ± ± ± ± ± ± ±
0.7 2.2 0.5 1.1 0.8 0.6 2.1
30.33 58.50 64.80 50.40 55.30 55.65 63.10
1:6 ± ± ± ± ± ± ±
1.5 0.7 1.4 2.2 1.3 2.3 1.2
31.35 55.85 53.85 52.25 45.70 51.15 53.00
± ± ± ± ± ± ±
1.3 2.3 1.1 1.8 0.8 2.1 1.5
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more favorable to carry out transesterification using whole-cell biocatalyst. Following other studies as well are in accordance with the above justifications. Guldhe et al. [34] has reported synthesis of biodiesel from microalgae (S. obliquus) using immobilized Aspergillus niger whole cell lipase biocatalyst while taking methanol to oil molar ratio of 3:1, 5:1 and 7:1. After optimization, 5:1 M ratio was reported to be optimum amount. In another study, Amoah et al. [35] reported addition of methanol with oil at 1:1 M ratio and further increasing upto a molar ratio of 4:1 (methanol to oil) using Aspergillus oryzae as whole cell catalyst. Almyasheva et al. [36] has also reported stepwise addition of methanol with four molar equivalent (1 M equivalent of methanol in 1st, 2nd, 3rd, & 4th phases) usig Aspergillus niger as whole cell biocatalyst.
Table 2 Different parameters of ethyl-propyl and ethyl-butyl esters produced from cottonseed oil. Parameters
Unit
Ethylpropyl
Ethylbutyl
ASTM D6751 standard for biodiesel
Gross calorific Value (GCV) Flash Point Ash Viscosity @ 40 °C Sediment (%) Sulphur (%)
Kcal/kg
8866
9382
7870
°C (minimum) % w/w centipoise % w/w % w/w (maximum) gm/cc °C °C %
104 ND 6.02 ND ND
71 ND 7.11 ND ND
130 NA 4.0–6.0 0.05 0.05
0.87 −5 −20 91
0.86 −6 −26 90
0.89 NA −15.0 to 10.0 NA
Density @15 °C Cloud point Pour point Ester Content
3.3. Effect of increase in the carbon chain Esters obtained from blends of different alcohols (Table 1), the yield of ethyl ester was lower when compared to their corresponding alkyl ester except for methanol. This clearly indicates that the alcohol with longer carbon chain is preferred over the shorter carbon chain, during the transesterification process, an observation that is notable in the present study. There was a significant (p < 0.05) variation between the yields of ethyl ester over the yield of other alkyl esters in either of the molar ratio (Fig. 4). The present work also supports the previous observations [7,9,33], where unlike chemical catalysts, biocatalysts are more favourable for the synthesis of higher alcohol chain fatty acid esters. Generally, either methanol or ethanol is used as an acyl acceptor during transesterification, but these alcohols are soluble in water and thus inactivate the lipase [37,38]. The solubility of methanol and ethanol in oil is approximately 50% and 75% respectively [39]. The solubility of propanol and other higher alcohols is comparatively higher as they are completely miscible with vegetable oils because of their low polarity compared to methanol and ethanol [40]. As a result, transesterification reactions with alcohols having three or more carbon atoms are monophasic throughout which enhances the rate and extent of the reaction [41,42]. Unlike methanol and ethanol, there are no mass transfer limitations across interfaces between phases in the case of higher alcohols, since all reactants are in a single (oil) phase.
NA: Not available, ND: Not detected.
characteristics of esters obtained from ethanol-propanol and ethanolbutanol were approximately similar as per the standards specified in Table 2. 3.5. Effect on cloud point and pour point Pour point (PP) and cloud point (CP) have been used to estimate the behaviour of diesel fuels in cold weather and lower PP and CP values exhibit improved cold flow properties of biodiesel. In present work, CP and PP of ethyl-propyl and ethyl-butyl ester blends are presented in Table 2. The observed CP and PP of ethyl-butyl are lower than ethylpropyl ester. This indicates that with an increase in the chain length of alcohol portion of fatty acid esters, there is a concomitant decrease in their crystallization temperature. The findings are also supported by the previous report wherein the CP of methyl, ethyl, isopropyl, and 2-butyl esters sourced from soybean oil were −2, −2, −9 and −12 °C, respectively [44] whereas CP of beef tallow sourced methyl, ethyl, propyl, and butyl esters was 17 °C, 15 °C, 12 °C, and 9 °C, respectively [45]. Similarly, blends of higher chain alcohols like butanol, pentanol and hexanol with fatty acid methyl ester generated from waste cooking oil have been shown improved cold flow properties [46]. Other than ester head group, cold flow properties also depend upon the double bond content of oil used for transesterification. Saturated fatty acids exhibit high temperature of crystallization and thus produce biodiesel with inferior cold flow properties. Soybean oil contains 14% saturated fatty acids and beef tallow contains 25% saturated fatty acids. Even though, cottonseed oil contains 26% saturated fatty acids, the CP of ethyl-propyl (−5) and ethyl-butyl ester (−6) was found to be better than soybean oil and beef tallow. Similarly, PP of biodiesel also depends upon the degree of saturation and ester head group [47]. In the current work, the PP values of ethylpropyl and ethyl-butyl esters were −20 °C and −26 °C, which is better, in spite of the presence of 26% saturated fatty acids.
3.4. Effect of blends on fuel properties of ethyl-propyl and ethyl-butyl ester In general practice, alcohol-biodiesel blends are used to overcome the disadvantage of higher viscosity, poor cold flow behaviour for biodiesel and the lower cetane number for alcohols; as the blends observably show better fuel properties compared to biodiesel fuel [43]. After transesterification for a selected ratio of alcohol, a higher ester yield was obtained from ethanol-propanol (91%) and ethanol-butanol (90%) blends. Biodiesel obtained after synthesis were examined for gross calorific value (GCV), flash point, ash, kinematic viscosity, sediment, sulfur content, density, cloud point (CP) and pour point (PP). The
3.6. Effect on flashpoint The flashpoint is the lowest temperature at which the fuel can form an ignitable mixture with air. The flashpoints of ethyl-propyl and ethylbutyl esters were 104 °C and 71 °C, respectively, which are higher than that of diesel fuel, which is 60 °C. This difference is related to the components present in each fuel. Fossil diesel is composed of low molecular weight molecules and a branched compound that leads to the reduction of the flash point. In contrast, among biodiesels the difference in flash point possibly depends upon fatty acid chain length, level of unsaturation, types of alcohol used and residual alcohol present after transesterification. According to ASTM D6751 standard, minimum flash
Fig. 4. Variations in the yields of ethyl ester (EE) and other alkyl esters (other) in both 1:4 and 1:6 M ratios. 5
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well as blends of ethanol-long chain alcohols. Cold flow properties of ethyl-alkyl ester blends were better than properties of standard biodiesel. Alkyl esters with larger ester head groups (such as ethyl, propyl, or butyl) have greater energy content. The viscosity of the ethyl-propyl and ethyl-butyl esters was found to be higher than stearic acid ethyl ester. Also, flash point got improved using alcohol blends compared to standard biodiesel.
point of 100% methyl ester should be 130 °C. In present work, reduction in flash point from ethyl-propyl to ethyl-butyl clearly indicated that alcohol chain length significantly reduce the flash point of biodiesel. A higher flash point indicates that the biodiesel is less flammable than petroleum diesel; hence, biodiesel is safer to handle [48]. 3.7. Effect on viscosity and density
Acknowledgments
Among the biodiesel properties, viscosity and density are the most important parameters that affect the engine performance and the emission characteristics. Higher viscosity affects the fuel atomization during injection into the combustion chamber and increases the carbon deposition on fuel filter, demands more energy from the fuel pump [49]. In the present study, ethyl-propyl and ethyl-butyl esters had viscosities of 6.02 cP and 7.11 cP respectively at 40 °C (Table 2). Factors such as larger ester head group and longer chain length result in high kinematic viscosity. The kinematic viscosity of methyl esters of lauric, myristic, palmitic, and stearic acids is 2.43, 3.30, 4.38, and 5.85 cP respectively and kinematic viscosities of methyl, ethyl, and butyl esters of stearic acid is 5.85, 5.92, and 7.59 cP respectively [5]. In our findings, the viscosity of the ethyl-propyl and ethyl-butyl esters was found to be higher when compared to the stearic acid ethyl ester (5.92). This indicates that a larger head group increases the viscosity of biodiesel. The density of ethyl-propyl and ethyl-butyl ester blends was observed to be 0.87 gm/cc and 0.86 gm/cc which fulfill the prescribed limits. Biodiesel has densities between 0.860 gm/cc and 0.897 gm/cc at 15 °C which is higher than that of petroleum diesel (0.83 gm/cc) [50].
The authors acknowledge SAI Labs, Thapar Institute of Engineering and Technology, Patiala, India for NMR facility. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fuel.2019.116258. References [1] Ranganathan SV, Narasimhan SL, Muthukumar K. An overview of enzymatic production of biodiesel. Bioresour Technol 2008;99(10):3975–81. [2] Edith O, Janius RB, Yunus R. Factors affecting the cold flow behaviour of biodiesel and methods for improvement—a review. Pertanika J Sci Technol 2002;20(1):1–14. [3] Prabu A, Anand RB. Production and application of biodiesel–a case study. Int J Eng Res Develp 2012;2(2):28–41. [4] Alptekin E, Canakci M. Determination of the density and the viscosities of biodiesel–diesel fuel blends. Renew Energy 2008;33(12):2623–30. [5] Moser BR. Biodiesel production, properties, and feedstocks. Vitro Cell Devpl 2009;45(3):229–66. [6] Du W, Li W, Sun T, Chen X, Liu D. Perspectives for biotechnological production of biodiesel and impacts. Appl Microbiol Biotechnol 2008;79(3):331–7. [7] Hossain ABMS, Boyce AN, Salleh A, Chandran S. Impacts of alcohol type, ratio and stirring time on the biodiesel production from waste canola oil. Afr J Agric Res 2010;5(14):1851–9. [8] Zhou W, Konar SK, Boocock DG. Ethyl esters from the single-phase base-catalyzed ethanolysis of vegetable oils. J Am Oil Chem Soc 2003;80(4):367–71. [9] Chowdary GV, Prapulla SG. Enzymatic synthesis of ethyl hexanoate by transesterification. Int J Food Sci Technol 2003;38(2):127–33. [10] Malcata FX, Reyes HR, Garcia HS, Hill Jr CG, Amundson CH. Kinetics and mechanisms of reactions catalysed by immobilized lipases. Enzyme Microb Technol 1992;14(6):426–46. [11] Ban K, Kaieda M, Matsumoto T, Kondo A, Fukuda H. Whole cell biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized within biomass support particles. Biochem Eng J 2001;8(1):39–43. [12] Bacovsky D, Körbitz W, Mittelbach M, Wörgetter M. Biodiesel production: technologies and European providers. IEA Task 2007;39:9. [13] Ghaly AE, Dave D, Brooks MS, Budge S. Production of biodiesel by enzymatic transesterification. Am J Biochem Biotechnol 2010;6(2):54–76. [14] Kato M, Fuchimoto J, Tanino T, Kondo A, Fukuda H, Ueda M. Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl Microbiol Biotechnol 2007;75(3):549–55. [15] Robles-Medina A, González-Moreno PA, Esteban-Cerdán L, Molina-Grima E. Biocatalysis: towards ever greener biodiesel production. Biotechnol Adv 2009;27(4):398–408. [16] Aulakh SS, Prakash R. Optimization of medium and process parameters for the production of lipase from an oil-tolerant Aspergillus sp. (RBD-01). J Basic Microbiol 2010;50(1):37–42. [17] Prakash R, Aulakh SS. Transesterification of used edible and non-edible oils to alkyl esters by Aspergillus sp. as a whole cell catalyst. J Basic Microbiol 2011;51(6):607–13. [18] Samukawa T, Kaieda M, Matsumoto T, Ban K, Kondo A, Shimada Y, et al. Pretreatment of immobilized Candida antarctica lipase for biodiesel fuel production from plant oil. J Biosci Bioeng 2000;90(2):180–3. [19] Silva-Santisteban BOY, Filho FM. Department of Food Engineering, FEA-University of Campinas, Campinas, SP, CEP 13083–970, CP 6121, Brazil. Enzyme Microb Technol 2005;36(5–6):717–24. [20] Sharma A, Verma A, Luxami V, Melo JS, D’Souza SF, Prakash NT, et al. New proton nuclear magnetic resonance-based derivation for quantification of alkyl esters generated using biocatalysis. Energy Fuel 2013;27(5):2660–4. [21] Gelbard G, Bres O, Vargas RM, Vielfaure F, Schuchardt UF. 1H nuclear magnetic resonance determination of the yield of the transesterification of rapeseed oil with methanol. J Am Oil Chem Soc 1995;72(10):1239–41. [22] Georgogianni KG, Kontominas MG, Pomonis PJ, Avlonitis D, Gergis V. Conventional and in situ transesterification of sunflower seed oil for the production of biodiesel. Fuel Process Technol 2008;89(5):503–9. [23] Meher LC, Sagar DV, Naik SN. Technical aspects of biodiesel production by transesterification—a review. Renew Sustain Energy Rev 2006;10(3):248–68. [24] Vyas AP, Verma JL, Subrahmanyam N. A review on FAME production processes.
3.8. Effect on gross calorific value The gross calorific value (GCV) is the total energy released as heat when a fuel undergoes complete combustion with oxygen [51]. In the present study, the GCV of ethyl-propyl and ethyl-butyl ester blends were 8866 and 9382 Kcal/kg respectively which is higher than standard biodiesel (7870 Kcal/kg) (Table 2). The main factor that influences the calorific value or energy content of biodiesel is carbon to oxygen ratio which is directly proportional to chain length [5,49]. Biodiesel fuels with larger ester head groups (such as ethyl, propyl, or butyl) have greater energy content as a result of their greater carbon to oxygen ratio. The present study, thus, demonstrates that higher ester yield can be achieved using a molar ratio of 1:4 or 1:6 (oil: alcohol) and whole-cell fungal system as a biocatalyst for the production of fatty acid alkyl esters. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Biodiesel, thus produced, also showed better cold flow properties with increase in a carbon chain of alcohol. Alcohol other than ethanol facilitates the only marginal increase in ester yield with an increase in the carbon chain. 4. Conclusion The present study investigated production of biodiesel form refined cottonseed oil using Aspergillus sp. as whole-cell biocatalyst, and mixture of ethanol and other alcohols (methanol to octanol) as acyl acceptors. The effect of alcohols blends in two different ratios of oil to alcohol viz., 1:4 and 1:6, on ester yield and its fuel properties was evaluated. The results showed no significant difference in the ester yield with variation in the molar ratio of oil to alcohol from 1:4 to 1:6. Ester yield up to 91% and 87% was obtained using 1:4 and 1:6 M ratio respectively. By the use of a lower molar ratio (1:4) and whole-cell catalyst, a significant reduction in the cost of production of biodiesel is as expected. Alcohol other than ethanol facilitated only a marginal increase in ester yield with no significant variation from propanol to octanol. Longer chain alcohol was preferred over the shorter chain as evident by increasing yield of ester in the methanol-ethanol blend as 6
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