Full Text

Turn on search term navigation

Nde et al. AMB Expr (2015) 5:83 DOI 10.1186/s13568-015-0172-x

ORIGINAL ARTICLE

Open Access

http://crossmark.crossref.org/dialog/?doi=10.1186/s13568-015-0172-x&domain=pdf

Web End = ESolvent-free, enzyme-catalyzedbiodiesel production frommango, neem, andshea oils viaresponse surface methodology

http://crossmark.crossref.org/dialog/?doi=10.1186/s13568-015-0172-x&domain=pdf

Web End = http://crossmark.crossref.org/dialog/?doi=10.1186/s13568-015-0172-x&domain=pdf

Web End = Divine Bup Nde1,2*, Carlos Astete1 and Dorin Boldor1

Abstract

Mango, neem and shea kernels produce non-conventional oils whose potentials are not fully exploited. To give an added value to these oils, they were transesteried into biodiesel in a solvent-free system using immobilized enzyme lipozyme from Mucor miehei. The Doehlert experimental design was used to evaluate the methyl ester (ME) yieldsas inuenced by enzyme concentrationEC, temperatureT, added water contentAWC, and reaction timeRT. Biodiesel yields were quantied by 1H NMR spectroscopy and subsequently modeled by a second order polynomial equation with interactions. Lipozyme enzymes were more tolerant to high temperatures in neem and shea oils reaction media compared to that of mango oil. The optimum reaction conditions EC, T, AWC, and RT assuring near complete conversion were as follows: mango oil 7.25 %, 36.6 C, 10.9 %, 36.4 h; neem oil EC = 7.19 %, T = 45.7 C,

AWC = 8.43 %, RT = 25.08 h; and shea oil EC = 4.43 %, T = 45.65 C, AWC = 6.21 % and RT = 25.08 h. Validation

experiments of these optimum conditions gave ME yields of 98.1 1.0, 98.5 1.6 and 99.3 0.4 % for mango, neem

and shea oils, respectively, which all met ASTM biodiesel standards.

Keywords: Biodiesel, Enzyme, Mango, Methyl esters, Neem, Shea

Introduction

Over the last decades, the importance of biodiesel has grown steadily as its research moved solidly in the commercialization arena (Howell 2009). Among its advantages over conventional diesel fuels one can include the facts that biodiesel is biodegradable, renewable and produces low levels of CO2 which make it environmentally friendly fuel. However, biodiesel fuel does not compete favorably economically with conventional diesel due to the high cost of vegetable or animal oils, the principal raw material in biodiesel manufacture, and the lack of government subventions in most countries as it is the case with conventional diesel. Given the upward trends in the price of conventional diesel fuels and considering its exhaustive nature, it is feared that in the near future conventional diesel may become expensive and/or

completely exhausted. There is therefore a critical need to search for suitable alternatives such as biodiesel to complement future fuel needs.

One feature of the transesterication reaction between the oils and short chain alcohol into biodiesel is the use of a catalyst to increase reaction rates and conversion yields. To this eect chemical catalysis and enzymatic catalysis have been used extensively in biodiesel research (Ma and Hanna 1999). Though the use of chemical catalysis in transesterication reactions is efficient in terms of reaction time, there are several setbacks such as difficulty in the recovery and purication of glycerol byproduct and the energy-intensive nature of the process, non-reusable nature of the recovered homogenous catalyst and soap formation (Sha et al. 2003). Enzymatic catalysis on its part is time consuming and the high cost of enzymes make the reaction very expensive. Enzymatic catalysis however has several advantages such as easy separation of by-products, synthesis of specic alky esters, and transesterication of glycerides with high free fatty acid content which eliminates the need for a two-step reaction

*Correspondence: [email protected]

2 Present Address: Department of Food and Bio-resource Technology, College of Technology, University of Bamenda, P.O. Box 39, Bamenda, CameroonFull list of author information is available at the end of the article

2015 Nde et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Nde et al. AMB Expr (2015) 5:83

Page 2 of 12

process (Nelson et al. 1996). Several researchers have shown that the use of immobilized enzymes can signicantly reduce biodiesel production cost because of the reusability of the immobilized enzymes (Dizge and Keskinler 2008; Shah and Gupta 2007). The use of enzymatic catalysis in biodiesel synthesis therefore remains a viable alternative.

So far, each country develops biodiesel feedstock according to its national conditions. For example, the United States mainly uses genetically modied soybean oil, the European Union and Canada use rapeseed oil while palm oil has been used in Malaysia and Indonesia to produce biodiesel due to the relative abundance of these raw materials in the respective countries (Tan etal. 2010). Other promising sources include non-edible oils such as jatropha (Tiwari etal. 2007; Sahoo and Das 2009), and microalgae and microbial oils some of which have short production cycles and can be produced by fermentation using inexpensive sources, such as CO2 or waste water (Mata etal. 2010; Pokoo-Aikins etal. 2010; Schenk et al. 2008). It is therefore necessary that each country or region evaluate the potentials of its available lipid sources for the production of biodiesel. The potentials of mango, neem and shea kernel oils which abound in many (sub)tropical regions of the world for the production of biodiesel fuel have not received much attention from researchers.

The mango tree is grown mostly for its fruits, which contain a sweet pulp, in about 90 countries and is rated among the top 20 agricultural products of the world (FAOSTAT 2013). When mature and ripe, the pulp is mostly eaten in the fresh state as a snack. The peels and the nuts are considered as waste and are thrown away after the eshy pulp has been consumed.

The neem tree is adapted to hot and dry climates, and is commonly planted in arid and semi-arid areas found in about 78 countries world-wide and is used in a further nine (Frster and Moser 2000). It grows both within its natural range (South Asia) as well as in Sub Saharan Africa, Central and Southern America, the Caribbean, Philippines, and the Middle East.

The shea tree grows mainly in tropical Africa and is today the second most important oil crop in Africa after the palm nut tree, though its potential is not fully exploited. Published data (Maranz et al. 2003) state that at least 500 million production trees are accessible in West Africa, which equates to a total of 2.5 million tonnes of dry kernel per annum (based on 5kg dry kernel per tree).

Mango (Lakshminarayana et al. 1983), neem (Kaura et al. 1998) and shea kernels contain 715, 1442 and 3555% (Bup etal. 2011; Honfo etal. 2013) oils indicating their potentials as a vegetable oil source. While neem

oil is not eaten in the production areas, mango and shea oils have been used as cocoa butter equivalents in chocolates and margarine formulations. All three types of oils nd use in traditional medicines and cosmetic formulations (Bup etal. 2011; Frster and Moser 2000; Lakshminarayana etal. 1983). However, reports have shown that potential for the production of these non-conventional oils are still largely underexploited. For example only 45% of total shea kernels produced annually are actually collected and processed (Holtzman 2004). The amounts of neem and mango kernels processed yearly are far less than shea nuts. Consequently it can be stated that these kernels often rot away during the production season and represent instead an environmental concern. At present there are no modern factories in African countries for the processing of mango and neem kernels into oil, which may be due partly to the fact that market demands from the existing users are not strong enough to encourage production. A diversication of the uses of these oils for the production of biodiesel may be a correct measure to encourage their exploitation, which will have important positive eects on the economies of the processors and the countries producing them. Studies on the enzymatic transesterication of these oils, as far as we know, do not exist in the literature.

Response surface methodology has been used by many researchers to establish optimum production conditions for biodiesel (Jeong and Park 2009; Shieh et al. 2003) because of the many advantages the method presents. The objective of this work therefore is to report the lipozyme TL IM catalyzed transesterication yields for biodiesel production from neem, shea and mango oils using response surface methodology and the Doehlert design. The study also reports for the rst time 1H NMR spectra for mango neem and shea oils.

Materials andmethods

Materials

Immobilized lipase Lipozyme TL IM from Rhizomucor miehei and 99 % methanol used in the experiments were bought from Sigma Aldrich. Ultra-pure water used in the analysis was produced in the laboratory using a Barnstead NANOpure Diamond (Thermo SCIENTIFIC) apparatus. Neem and shea oils were bought from local processors in North Cameroon while mango oil was bought from http://www.Amazon.com

Web End =http://www.Amazon.com . The fatty acid proles and some physico-chemical properties of the oils used in the analysis are presented in Table1.

Experimental design

Response surface methodology and the Doehlerts experimental design requiring 42 experiments (21 experiments in duplicate) was used to study the enzyme catalyzed

Nde et al. AMB Expr (2015) 5:83

Page 3 of 12

Table 1 Fatty acid composition, acid value andmoisture content ofthe oils used forenzymatic transesterication

Fatty acid composition (%) Physical properties

C16:0 C18:0 C18:1 C18:2 SFA USFA Acid value Moisture content

Mango 15.6 30.2 48.2 6.0 45.8 54.2 1.0 0.1 0.085

Neem 19.6 7.8 51.8 20.8 27.4 72.6 2.4 0.3 0.082

Shea 5.7 41.8 45.6 6.9 47.5 52.5 15.2 0.3 0.230

trans-esterication of the three oils into biodiesel. The factors and ranges of the parameters studied were enzyme concentration (310 wt% of oil mass), temperature (3060C), quantity of added water (515 wt% of oil mass) and time (1236h). The solvent/oil mole ratio and the agitation speed were maintained constant at 3:1 and 200rpm, respectively. The Doehlert matrix used in this study is presented in Table2.

Experimental procedure ofthe transesterication process

An incubator shaker (C25K New Brunswick Scientic, USA) was used in this study. In each experimental run 5g of the oil was weighed into a 125ml conical ask and the calculated amount of enzyme, methanol and water based on the weight of the oil were added in succession to the reaction ask. These were then tightly corked and placed into the incubator shaker and the reaction allowed to run at the required temperature and time as shown in Table 2. From preliminary experiments, addition of 3:1 methanol-oil molar ratio in a single step yielded only about 5 % FAME. Subsequently, methanol was added in three steps to the reaction mixture, with 1/3 of the required amount at 0, 3 and 6h to achieve a total mole ratio of 3:1. Preliminary experiments (Fig. 1) equally showed that in 2h almost the entire methanol added had reacted so 3h addition interval was chosen to ensure a complete reaction. In a solvent system (hexane), reactions yield were lower than those obtained in a solvent free system, therefore all the reactions were carried out in a solvent free system. At the end of each experiment the mixture was ltered through a Whatman lter paper. Samples were then collected and the biodiesel yield analysed by 1H NMR spectroscopy (Jin etal. 2007). A total of 42 experiments (21 experiments in duplicates) were carried out for each oil, following the experimental plan based on the Doehlerts experimental design.

Analysis ofbiodiesel yield by1H NMR spectroscopy

Samples for 1H NMR spectroscopy were prepared by dissolving 0.020.05g of biodiesel produced in 1ml of chloroform. 1H NMR spectra were obtained using a BRUKER 500 MHz AVANCE III instrument with CDCl3 as solvent and TMS as an internal standard. 1H spectra were

recorded with pulse duration of 45C and 16 scans, following the procedure described by Gelbard etal. (1995). The percentage conversion (Y) of oil to biodiesel was calculated from the relation (Gelbard et al. 1995; Suganya etal. 2014):

ACH3 is the integration value of the methoxy protons of the ME (the strong singlet) and ACH2 the integration value of the methylene protons. Factors 2 and 3 were derived from the fact that, the methylene carbon possesses two protons, while the alcohol (methanol derived) carbon has three attached protons.

Statistical analysis

A second order polynomial with interactions represented by Eq.2 was selected for the analysis. b0, bi, bii and bij are model coefficients for intercept, linear, quadratic and interaction terms, respectively. Coefficients of the model were determined through multiple linear regression analysis using Sigmaplot 12.5 (Systat Software Inc, San Jose, USA).

To optimize the cooking process, the optimum point of Eq.2 was dened as the point where the rst partial derivative of the function equals zero: That is

The system of equations for each response was then solved using the matrix method using Microsoft Excel (Version 10, Microsoft Corp., Redmond, WA, USA).

Validation ofmodels

Two criteria, the regression coefficient (R2) and the percentage Absolute Error of Deviation (AED) between experimental and calculated results were used to evaluate

Y = 2ACH3 3ACH2

(1)

y = b0 +

[notdef]

i=1

bixi +

3 biix2i +

[notdef]

i=1

[notdef]

j=i+1

bijxixj

(2)

~y/~x1 = b1 + 2b11x1 + b12x2 + b13x3

~y/~x2 = b2 + 2b12x1 + b22x2 + b23x3

~y/~x3 = b3 + 2b13x1 + b23x2 + b33x3

= 0

(3)

Nde et al. AMB Expr (2015) 5:83

Table 2 Experimental design andconversion yields ofmango, neem andshea oils

Page 4 of 12

S/no Experimental matrix Responses

Coded values Real values Conversion yield (%)

x1 x2 x3 x4 X1 X2 X3 X4 Neem Mango Shea

1 1 0 0 0 10.00 45.00 10.00 24.00 91.68 92.27 98.77 1 1 0 0 0 10.00 45.00 10.00 24.00 92.71 91.11 99.70 2 1 0 0 0 3.00 45.00 10.00 24.00 90.51 56.24 58.23

2 1 0 0 0 3.00 45.00 10.00 24.00 90.77 61.69 60.72

3 0.5 0.866 0 0 8.25 60.00 10.00 24.00 71.74 63.87 99.03 3 0.5 0.866 0 0 8.25 60.00 10.00 24.00 68.98 68.45 99.31 4 0.5 0.866 0 0 4.75 30.00 10.00 24.00 94.76 82.69 86.03

4 0.5 0.866 0 0 4.75 30.00 10.00 24.00 96.24 85.15 84.15

5 0.5 0.866 0 0 8.25 30.00 10.00 24.00 93.64 91.38 95.48

5 0.5 0.866 0 0 8.25 30.00 10.00 24.00 94.34 89.37 94.98

6 0.5 0.866 0 0 4.75 60.00 10.00 24.00 82.41 48.39 72.86

6 0.5 0.866 0 0 4.75 60.00 10.00 24.00 86.94 49.38 73.74

7 0.5 0.289 0.816 0 8.25 50.01 15.00 24.00 92.54 91.01 82.70 7 0.5 0.289 0.816 0 8.25 50.01 15.00 24.00 92.53 89.77 85.19 8 0.5 0.289 0.816 0 4.75 39.99 5.00 24.00 89.29 68.70 94.55

8 0.5 0.289 0.816 0 4.75 39.99 5.00 24.00 85.71 70.11 94.70

9 0.5 0.289 0.816 0 8.25 39.99 5.00 24.00 93.33 90.82 96.61

9 0.5 0.289 0.816 0 8.25 39.99 5.00 24.00 92.52 90.88 97.11

10 0 0.577 0.816 0 6.50 54.99 5.00 24.00 89.82 77.04 97.07

10 0 0.577 0.816 0 6.50 54.99 5.00 24.00 88.52 73.64 97.70

11 0.5 0.289 0.816 0 4.75 50.01 15.00 24.00 97.17 84.99 60.62

11 0.5 0.289 0.816 0 4.75 50.01 15.00 24.00 93.58 75.32 59.74

12 0 0.577 0.816 0 6.50 35.01 15.00 24.00 98.68 93.14 81.77

12 0 0.577 0.816 0 6.50 35.01 15.00 24.00 97.65 92.52 78.24

13 0.5 0.289 0.204 0.791 8.25 50.01 11.25 36.00 97.88 96.30 100.00 13 0.5 0.289 0.204 0.791 8.25 50.01 11.25 36.00 98.90 98.36 99.89 14 0.5 0.289 0.204 0.791 4.75 39.99 8.75 12.00 93.44 45.32 66.50

14 0.5 0.289 0.204 0.791 4.75 39.99 8.75 12.00 95.30 47.85 62.75

15 0.5 0.289 0.204 0.791 8.25 39.99 8.75 12.00 95.24 58.58 92.99

15 0.5 0.289 0.204 0.791 8.25 39.99 8.75 12.00 95.46 57.11 97.78

16 0 0.577 0.204 0.791 6.50 54.99 8.75 12.00 95.58 71.97 99.78

16 0 0.577 0.204 0.791 6.50 54.99 8.75 12.00 95.63 71.15 95.09

18 0.5 0.289 0.204 0.791 4.75 50.01 11.25 36.00 99.58 88.42 99.65

18 0.5 0.289 0.204 0.791 4.75 50.01 11.25 36.00 98.52 87.77 99.85

19 0 0.577 0.204 0.791 6.50 35.01 11.25 36.00 99.79 96.24 99.61

19 0 0.577 0.204 0.791 6.50 35.01 11.25 36.00 99.80 96.74 99.54

20 0 0 0.612 0.791 6.50 45.00 6.25 36.00 98.39 97.14 99.54

20 0 0 0.612 0.791 6.50 45.00 6.25 36.00 95.54 96.33 99.54

21 0 0 0 0 6.50 45.00 10.00 24.00 90.70 97.34 96.62 21 0 0 0 0 6.50 45.00 10.00 24.00 91.43 93.58 98.22 R2 0.808 0.882 0.949 AED (%)\ 2.58 5.95 2.85

X1 enzyme concentration, X2 reaction temperature, X3 quantity of added water and X2 reaction time

Nde et al. AMB Expr (2015) 5:83

Page 5 of 12

110

100

Conversion yield (%)

Mango Neem Shea

Constant conditions: T = 45 C, AWC = 10%, RT = 24 h

60 3 4 5 6 7 8 9 10

65 30 35 40 45 50 55 60

Enzyme concentration (wt %)

Temperature (oC)

105

110

100

Conversion yield (%)

Mango Neem Shea

75 5 6 7 8 9 10 11 12 13 14 15 12 15 18 21 24 27 30 33 36

Quantity of added water (wt %)

Time (h)

Fig. 1 a Inuence of enzyme concentration on methyl ester yields of the three oils at constant temperature (45 C), quantity of added water (10 %) and reaction time (24 h). b Inuence of enzyme concentration (top) and temperature (bottom) on methyl ester yields of the three oils when the other parameters are maintained constant quantity of added enzyme concentration (6.5 %) water (10 %) and reaction time (24 h). c Inuence of quantity of added water on methyl ester yields of the three oils at constant enzyme concentration (6.5 %), temperature (45 C) and reaction time (24 h). d Inuence of quantity of reaction time on methyl ester yields of the three oils at constant enzyme concentration (6.5 %), temperature (45 C) and quantity of added water (10 %)

the validity of the models. A model was considered valid if R2>0.7 and/or AED<10% (Bup etal. 2012). Regression coefficients were obtained from multiple linear regression analysis carried out on the results using SigmaPlot 12.5 software while AED was calculated from Eq.4.

where Yexp and Ymod are the values obtained from experiments and from the model, respectively. p is the number of points at which measurements were carried out.

Results

Modelling ofthe transesterication process

Validation conditions (R2>0.8 and AED<10%) for the selected second order models obtained from regression analysis were met for all the biodiesels (Table2). Analysis of variance showed that all the four parameters had significant influences (P<0.05) on the enzymatic transesterification process of mango, neem and shea oils into biodiesel (Table3). The validated models were therefore used to generate two dimensional and surface response plots to explain the main and the interaction effects of the factors on the biodiesel conversion yield.

AED (%) = 100

[notdef]

i=1

Yexp Y mod

Yexp

(4)

Nde et al. AMB Expr (2015) 5:83

Page 6 of 12

Table 3 Statistical analysis forthe Doehlert experimental design

Mango Neem Shea

Coe VMC P value Coe VMC P value Coe VMC P value

b0 95.46 37.27 <0.001 91.07 61.86 <0.001 97.42 38.57 <0.001 b1 14.14 5.52 <0.001 0.98 0.67 0.404 17.25 6.83 <0.001

b2 9.89 3.86 <0.001 6.61 4.49 <0.001 0.04 0.02 0.973

b3 5.68 2.22 0.029 3.37 2.29 0.015 13.22 5.24 <0.000

b4 20.86 8.14 <0.001 1.64 1.12 0.244 12.22 4.84 <0.001 b11 20.13 7.86 0.003 0.35 0.24 0.913 18.07 7.15 <0.001

b22 24.12 9.42 <0.001 6.70 4.55 0.044 6.27 2.48 0.076

b33 7.39 2.89 0.219 3.91 2.65 0.216 11.81 4.68 0.001

b44 17.32 6.76 0.008 10.37 7.05 0.003 4.58 1.82 0.186

b12 6.25 2.44 0.281 7.40 5.03 0.02 9.08 3.60 0.009

b13 9.08 3.55 0.165 2.45 1.66 0.471 9.97 3.95 0.01

b14 1.23 0.48 0.854 2.30 1.56 0.514 25.21 9.98 <0.001

b23 3.35 1.31 0.602 4.73 3.21 0.169 2.61 1.03 0.473

b24 17.45 6.81 0.014 2.68 1.82 0.447 7.54 2.99 0.053

b34 3.83 1.50 0.693 2.67 1.82 0.6 17.24 6.83 0.004

Table 4 ANOVA foryield

df SS MS F P value

Mango

Regression 14 9323.70 665.98 13.80 <0.001 Residual 25 1206.90 48.28Total 39 10,530.60Neem

Regression 14 1336.44 95.46 7.15 <0.001 Residual 25 333.99 13.36Total 39 1670.42Shea

Regression 14 7095.23 506.80 32.98 <0.001 Residual 25 384.13 15.37Total 39 7479.36

Eect ofindividual factors onthe biodiesel conversion yields ofmango, neem andshea oils

Plots of the eect of the individual factors on conversion yields were obtained from the validated equations by maintaining three of the factors constant at their central points and varying the other as a function of the biodiesel conversion. Generally conversion yields ranged from 45.3 to 98.4%, 69.0 to 99.8% and 58.2 to 100.0% for mango, neem and shea oils, respectively.

Eect ofenzyme concentration onbiodiesel conversion yield

Table3 shows that enzyme concentration had a signi-cant eect (p<0.05) on the biodiesel conversion yields of mango and shea oils. From Fig.1a it is observed that for mango and shea oils, biodiesel conversion yields increased from about 60 to 97 % and to almost complete conversion, respectively as enzyme concentration increased from 3 to about 7 %. Above 7 % of enzyme, the conversion yields decreased slightly to about 90 % for mango oil but remained almost constant for shea oil. From Table4, the contribution of enzyme concentration (b1+b11) to the value of the conversion yield for neem oil was less than 1% compared to about 14% for mango and shea oils, thus illustrating the non-signicant eect of this factor on the transesterication of neem oil.

Eect oftemperature onbiodiesel conversion yield

For mango oil, conversion yield increased from 85% at 30C to about 95 at 45C and then decreased steadily to about 65% at 60C, while for neem oil highest yields were

obtained at the beginning of the experiment (<35C) followed by a continuous decrease with an increase in reaction temperature (Fig. 1b). While maximum yields for mango and shea biodiesels were obtained at about 45C that of neem was at about 35C, indicating that enzyme activity may also depend on the type of oil involved in the transesterication process.

Eect ofquantity ofadded water onbiodiesel conversion yield

In this work the quantity of added water was investigated in the range 515% based on the weight of the oil taken for analysis. Conversion yields for all the 3 biodiesels

Nde et al. AMB Expr (2015) 5:83

were signicantly aected by the quantity of added water. When reaction temperature, time and enzyme concentrations were kept constant at the central point, conversion yields of mango and neem oils increased with the quantity of added water up to a certain value and then remained constant while that of shea oil decreased significantly as the quantity of added water was increased from 5 to 15% (Fig.1c).

Eect oftime onbiodiesel conversion yield

For mango and shea oils, conversion yields increased respectively from about 70 and 85% to almost complete conversion as reaction time increased from 12 to 36 h when all other factors were maintained constant at their center points. The linear eect of reaction time on neem biodiesel conversion yield was not signicant while the quadratic eect of reaction time was signicant explaining the small upward curvature observed on Fig.1d.

Interaction eect ofstudied factors onconversion yields ofmango, neem andshea oils

Surface response plots for the interaction eects were generated in SigmaPlot 12.5 to better visualize the combined eects of the factors on yield. The following section describes the interaction eects of two factors while the others were kept constant at the central point on the conversion yield. The interaction eect of the quantity of added water and temperature on yield was not signicant for the three biodiesels and has not been discussed.

Combined eect ofenzyme concentration andtemperature onconversion yield

Figure 2a gives the combined eect of enzyme concentration and temperature at constant quantity of added water (10%) and reaction time (24h). It was signicant (p < 0.05) for mango and shea oils but insignicant for neem oil. At low enzyme concentrations, the conversion yields varied only slightly with an increase in temperature for all the 3 oils. As enzyme concentration increased, conversion yields increased with temperature for both mango and shea oils to maximum values and then decreased again as enzyme concentration went above 8% at temperatures higher than 50C. The evolution of the conversion yield of neem oil remains fairly constant irrespective of the variation in temperature and enzyme concentration when the other factors are maintained constant.

Combined eect ofenzyme concentration andquantity ofadded water onconversion yield

At constant temperature (45C) and reaction time (24h) conversion yields decreased with the quantity of added water irrespective of the enzyme concentrations for

Page 7 of 12

shea oil but increased steadily with enzyme concentration especially at lower quantities of added water up to steady values (Fig.2b). For mango oil, conversion yields increased with the quantity of added water and with enzyme concentration up to steady values. The combined eect of temperature and enzyme concentration was insignicant on neem biodiesel yields; however, these conversions were very high (9098 %) throughout the experimental period under the explored conditions.

Combined eect ofenzyme concentration andreaction time onconversion yield

The variation of enzyme concentration and reaction time on conversion yield is shown in Fig.2c. At lower enzyme concentration, initial conversion yields for mango oil were greater than that of neem up to about 20 h reaction time. After 20 h that of shea oil was greater than that of mango oil. At high enzyme concentrations conversion yields increased with reaction time and attained steady values for both mango and shea oils. Conversion yields were higher for shea oils compared to mango and neem oils. Variation of conversion yields for neem oils under these conditions was not signicant. Maximum yields for shea oils were obtained at the highest enzyme concentration. At prolonged reaction times, conversion yields decreased probably due to aggregation of enzymes after long reaction times. In fact it was visually observed that enzymes aggregated and settled at the bottom of the reaction ask for most of the experimental runs that lasted more than 24h.

Combined eect oftemperature andreaction time onconversion yield

When the enzyme concentration and quantity of added water were held constant at their center points, only the variation of the conversion yield of mango oil was signi-cant (Table2). For mango oil, at temperatures lower than 40C, conversion yield increased from less than 50% to close to 100% as reaction time increased from 12 to 36h (Fig.2d). As temperatures increased above 40C, mango oil conversion yields decreased signicantly and were lowest at 60C. There was however no signicant dierence on shea and neem oil conversion yields at higher temperatures.

Combined eect ofquantity ofadded water andreaction time onconversion yield

Figure2e shows the variation of conversion yield for the 3 dierent oils as a function of quantity of added water and reaction time at constant enzyme concentration (6.5%) and reaction temperature (45C). The interaction eect of reaction time and quantity of added water on conversion yields of mango and neem oils was not signicant,

Nde et al. AMB Expr (2015) 5:83

Page 8 of 12

a b

110

100

Mango Neem Shea

100

Mango Neem Shea

ld(%)

(%)

ConversionYie

Conversionyield

Enzyme concentration (wt %)

Temperature (

oC)

Quantity of added water (wt %)

c d

Mango Neem Shea

120

110

Mango Neem Shea

100

(%)

ld(%)

Conversionyield

ConversionYie

Enzyme concentration (wt %)

55 o C)

Temperature (

Time

(h)

Time (min)

110

Mango Neem Shea

100

ld(%)

ConversionYie

Quantity of added water (wt %)

Time (h)

Fig. 2 a Combined eect of enzyme concentration and temperature on methyl ester yields of the three oils at constant quantity of added water (10 %) and reaction time (24 h). b Combined eect of enzyme concentration and quantity of added water on methyl ester yields of the three oils at constant temperature (45 C) and reaction time (24 h). c Combined eect of enzyme concentration and reaction time on methyl ester yields of the three oils at constant temperature (45 C) and quantity of added water (10 %). d Combined eect of temperature and reaction time on methyl ester yields of the three oils at constant enzyme concentration (6.5 %) and quantity of added water (10 %). e Combined eect of quantity of added water and reaction time on methyl ester yields of the three oils at constant enzyme concentration (6.5 %) and temperature (45 C)

Nde et al. AMB Expr (2015) 5:83

Page 9 of 12

contributing less than 2% to the value of the conversion yields of both biodiesels. On the other hand, the combined eect of the two factors (b34) had a signicant contribution to the conversion yield of shea oil. At shorter reaction times, conversion yields for shea oil decreased sharply with an increase in the quantity of added water and then remained fairly constant as reaction proceeded (Fig.2e). At quantities of added water greater than 10% conversion yields for shea oil again decreased sharply with prolonged reaction times. Though the combined eect of these factors had no signicant eect on mango yields, it can be observed from Fig. 2e that, at shorter reaction times, its conversion yields increased with an increase in the quantity of added water. This increase became more noticeable at longer reaction times. The statistically insignicant yet noticeable eect of the interaction eect of reaction time and temperature on the conversion yield of mango is due to the high contribution of the individual eect of reaction time (b4) which was greater than 8%. Note that b4 had a signicant eect on mango oil conversion yields. Again one notices the dierent behaviors of the dierent oils under similar reaction conditions.

Optimization ofthe enzymatic transesterication processes

Optimizations of the independent parameters gave the following optimum conditions: enzyme concentration 7.26 %, temperature 36.6 C, quantity of added water 10.9% and reaction time of 36.4 h for the production of mango oil ME. Corresponding values for neem and shea oils ME were 7.19 %, 45.65 C, 8.43 % and 25.08 h and 4.43 %, 45.65 C, 6.21 % and 25.08 h, respectively. ME yields calculated using the validated second order models showed that, under these conditions, a 100% conversion of the oils into biodiesel was achieved. Verication experiments conducted at these calculated optimum points reached conversion yields of 98.090.96, 98.481.62 and 99.250.35% for mango, neem and shea oils which were not signicantly dierent from the calculated yields, indicating that the developed models are adequate for use in describing enzymatic transesterication of these oils.

Discussion

Eect ofindividual factors onthe biodiesel conversion yields

Decrease in biodiesel conversion yields at high temperatures has been linked to the aggregation of enzymes at such temperatures. A similar observation was made in the trans-esterication of corn oil using lipozyme TL IM as catalyst (Wang et al. 2008). The variation of the conversion yield with enzyme concentration at constant temperature, time and quantity of added water, was

insignicant (P>0.05) for neem oil and varied only from 90 to 92% indicating that low enzyme concentrations can be used to achieve high biodiesel yields. This will reduce the cost of the production process.

Transesterication temperature had a signicant eect on the yields of mango and neem biodiesels but not on that of shea biodiesel. Reaction rate increases with temperature and reaction time due to the reduction of viscosity of the oil. This is favorable to increase the solubility of the oil in methanol and improve the contact between oil and methanol molecules, thereby reaching a better yield of ME (Suganya etal. 2013). Decrease in conversion yields after a certain temperature indicates the optimum temperature for enzyme activity after which the enzymes are denatured and can therefore not take part in the reaction. Several studies have indicated that in enzymatic catalysis each enzyme has an optimum temperature over which biodiesel yields are highest and this may depend on the type of oil used in the analysis (Bajaj etal. 2010; Dizge and Keskinler 2008).

Interaction eect offactors studied onconversion yields

For the combine eect of enzyme concentration and temperature on conversion yields it was observed that higher values of these parameters decrease conversion yields. The decrease in conversion yields above a certain enzyme concentration and temperature is attributed to the agglomeration and heat denaturation of the enzymes which both lower enzyme activity (Bajaj etal. 2010; Dizge and Keskinler 2008).

The presence of sufficient quantities of water in the reaction system can enhance the efficiency of lipase catalyzed reactions, while insufficient amounts of water in the reaction mixture can cause inactivation of lipase (Kaieda etal. 2001). Water addition to the reaction system may also reduce the resistance to mass transfer which results from the accumulation of glycerol produced during the reaction (Tran et al. 2012). However the amount of added water should be controlled as the presence of excess water also has negative consequences on the reaction yield. The quantity of added water should therefore be monitored carefully in order to determine the optimum quantity required for highest yields. According to Miller et al. (1988) and Yamane (1987), it is important to protect the water surrounding lipases for optimal conformation of the enzyme, and removal of the water can lead to both reversible, but mainly irreversible, changes in the protein structure. Previous studies have shown that the optimum quantity of added water may depend among other factors on the type of enzyme and the substrate (Kaieda etal. 2001). In this work it was observed that, in addition to the introduced factors (EC, T, AWC and RT) the quantity of water originally present

Nde et al. AMB Expr (2015) 5:83

Page 10 of 12

in the oil before transesterication may play an important role in the process. For example the initial quantity of water (0.229 0.02 %) present in shea oil was signicantly higher than that of mango (0.0820.01) and neem (0.0840.01) oils, respectively. This may explain the observed decrease of conversion yields on addition of water to shea oils, as addition of water above a certain level favors hydrolysis of the triglycerides instead of the transesterication reaction (Fjerbaek etal. 2009). At higher enzyme concentration (>8 %), the conversion yields remained fairly constant up to quantities of added water of 10%. When quantity of added water surpassed 10 % the conversion yield again decreased. We infer from this behavior that as the concentration of enzyme increases, the negative eect of water on conversion yield is overpowered by increased enzyme activity but with more and more water added to the system, enzyme activity is again reduced.

Considering the combined eect of temperature and reaction time the observed decreased conversion yields of mango oil at higher temperatures was linked to the denaturation of the enzymes at higher temperatures as earlier adduced. There was however no signicant dierence on neem and shea yields as a result of the interaction eect of reaction time and temperature even at higher temperatures. This dierence in the behavior of the conversion yields of the oils indicate that the lipozyme enzyme used in this work could be more tolerant to high temperatures in neem and shea oils compared to mango oils. It may be possible that mango oil may have some specic compound that may denature/deactivate the enzyme. This assertion requires further analysis. Again under these conditions shea conversion yields were for most of the times higher than those obtained with mango and neem oils.

Optimization

It was observed that optimum points for the transesterication of these oils under the same conditions differ between the oils. For example optimum operation temperature for mango oil was 36.6 C and therefore required a longer time of 36 h to attain complete conversion. Lipozyme was more tolerant to higher temperatures in neem and shea oils with a consequent reduction of the reaction time (25h) required to attain 100% conversion. Optimum conditions were reported elsewhere for biodiesel production using immobilized lipozymes from Mucor miehei for oil/ethanol molar ratio, temperature, added water content, and amount of enzyme of 1:3, 50C, 0% (vol/vol), and 0.4g of Lipozyme per 5.7mmol of sun ower oils, respectively (Selmi and Thomas 1998). Shieh etal. (2003) also reported that optimum synthesis conditions giving 92.2 % weight conversion of soybean

methyl esters using lipase from Mucor mieher were: reaction time 6.3h, temperature 36.5C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.

These results point to the fact that optimum conditions for transesterication vary with a variety of factors. In this work, since the three oils were processed under the same conditions using lipase from Mucor miehei, the differences observed in the behaviors of the yields of ME of the three dierent oils as seen in the preceding sections could be linked to one or a combination of composition, initial moisture content and the percentage of FFA present in the oil. Fatty acid compositions of the dierent oils used in the analysis are presented in Table1 which shows that neem oil is composed of more than 70% unsaturated fatty acids while shea and mango oils were composed of almost equal proportion of saturated and unsaturated fatty acids. The polyunsaturated nature of the neem oil can explain to a certain degree the reduced variation of the conversion yields of neem ME esters under most of the reaction conditions, since the oil remained in the liquid phase for the most part under these experimental conditions. In some cases especially at low temperatures, shea and mango oils existed rst in the solid phase before dissolution with the progression of the reaction and this probably could have led to the observed signicant variations of mango and shea biodiesels yields under the explained conditions.

Note that maximum yields were highest for shea oils compared to neem and mango oils. This was attributed to the high levels of free fatty acids (15.180.25%) in shea oil compared to 0.950.05 and 2.420.29% for mango and neem oils. Table2 clearly indicates that under the same conditions for most of the experiments the conversion yields varied in direct proportion with the acid value of the oil. That is, conversion yields were highest for shea, followed by neem and mango. One advantage of enzyme catalyzed transesterication reactions is that oils with high FFA can be easily converted to MEs without prior treatment as done in chemical catalysis, probably because no energy needs to be expended by the enzyme-based process to separate the FFA from the glycerol backbone (Lai etal. 2005).

Some quality parameters of the biodiesel determined following the ASTM methods (Table5) all met the ASTM biodiesel standards.

From the foregoing, it can be concluded that under the respective optimum conditions of enzyme concentration, temperature, quantity of added water and reaction time of 7.26%, 36.61C, 10.90% and 36.42h for mango oil, 7.19 %, 45.65 C, 8.43 % and 25.08 h for neem oil and 4.43%, 45.65C, 6.21% and 25.08h there was complete conversion of the oils into biodiesel. Under similar

Nde et al. AMB Expr (2015) 5:83

Page 11 of 12

Table 5 Properties offatty acid methyl esters ascompared tointernational standard requirements ofASTM

Property Units FAME value Required byASTM

Mango Neem Shea

Kinetic viscosity at 40 C mm2 s1 3.25 2.668 0.027 5.404 D 445 (1.96.0)

Density at 40 C kg m3 0.870 0.000 0.858 0.002 0.865 0.006

Acid value mgKOH g1 0.33 0.008 0.18 0.03 0.37 0.002 D 664 (Max. 0.5)

Cetane number 57.83 64.43 64.75 D 613 (47 minimum) Water content % volume 0.023 0.008 0.044 0.007 0.047 0.005 D 2709 (0.050 %)

Cloud point C 1718 1517 1920 ASTM D6751 (3 to 12)

Pour point C 13 12 13 ASTM D6751 (15 to 10)

conditions shea oil produced more biodiesel compared to mango and neem oils. Optimum processing conditions dier for each oil and therefore emphasize the need for such studies on each oilseed to establish precise parameters for appropriate scale up and industrial applications. The use of immobilized lipase from Mucor miehei is encouraged because apart from efficiently catalyzing the reaction, it is cheap and can be reused up to 10 times without signicant loss of activity (Krishna etal. 2001).

Author details

1 BAE Department, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, USA. 2 Present Address: Department of Food and Bio-resource Technology, College of Technology, University of Bamenda, P.O. Box 39, Bamenda, Cameroon.

Acknowledgements

Authors would like to acknowledge the nancial assistance provided by the US Department of State and The Fulbright Commission to Dr. Divine Nde Bup in the form of a Fulbright Scholar Award Commission (award # 68130893)as well as the Department of Biological and Agricultural Engineering at LSU AgCenter and LSU. The authors would like to thank Pranjali Muley, Mohamad Bareketi, Charles Henkel and Gustavo Aguilar for their assistance with the experimental and analytical portion of this study. Published with the approval of the Director of the Louisiana Agricultural Experiment Station as manuscript # 2014-232-16725.

Competing interests

The authors declare that they have no competing interests.

Received: 3 September 2015 Accepted: 11 December 2015

References

Bajaj A, Lohan P, Jha PN, Mehrotra R. Biodiesel production through lipase catalyzed transesterication: an overview. J Mol Catal B Enzym. 2010;62:914.

Bup DN, Abi C, Tenin D, Kapseu C, Tchiegang C. Optimisation of the cooking process of sheanut kernels (Vitellaria paradoxa Gaertn.) using the Doehlert Experimental Design. Food Biopro Technol. 2012;5:10817.

Bup DN, Kapseu C, Matos L, Mabiala B, Mouloungui Z. Inuence of physical pretreatments of sheanuts (Vitellaria paradoxa Gaertn.) on butter quality. Eur J Lipid Sci Technol. 2011;113:115260.

Dizge N, Keskinler B. Enzymatic production of biodiesel from canola oil using immobilized lipase. Biomass Bioenergy. 2008;32:12748.

FAOSTAT. http://faostat.fao.org/site/535/default

Web End =http://faostat.fao.org/site/535/default . 2013. Accessed 20 June 2014. Fjerbaek F, Christensen KV, Birgir N. A review of the current state of biodiesel production using enzymatic transesterication. Biotechnol Bioeng. 2009;102:1298395.

Frster P, Moser G. Status report on global Neem usage. Deutsche Gesellschaft fr Technische Zusammenarbeit (GTZ) Eschborn. 2000.

Gelbard G, Brs O, Vargas RM, Vielfaure F, Schuchardt UF. 1H nuclear magnetic resonance determination of the yield of the transesterication of rape-seed oil with methanol. JOACS. 1995;72:123941.

Holtzman J. The shea butter value chain. Study synthesis and recommendations for WATH. WATH Technical Report No. 1. USAID/West African Trade Hub (WATH): Accra; 2004.

Howell S. Industry needs, producers and distributors: Biodiesel. In: 2nd International Conference on Biofuels Standards and Measurements for Biofuels: Facilitating Global Trade. Brussels, Belgium; 2009.

Honfo FG, Linnemann AR, Akissoe N, Soumanou MM, van Boekel MAJS. Characteristics of traditionally processed shea kernels and butter. Inter J Food Sci Technol. 2013;48:171421.

Jeong G-T, Park D-H. Optimization of Biodiesel production from castor oil using response surface methodology. Appl Biochem Biotechnol. 2009;156:111.

Jin F, Kawasaki K, Kishida H, Tohji K, Moriya T, Enomoto H. NMR spectroscopic study on methanolysis reaction of vegetable oil. Fuel. 2007;86:12017.

Kaieda M, Samukawa T, Kondo A, Fukuda H. Eect of methanol and water contents on production of biodiesel fuel from plant oil catalyzed by various lipases in a solvent-free system. J Biosci Bioeng. 2001;91:125.

Kaura SK, Gupta SK, Chowdhury JB. Morphological and oil content variationin seeds of Azadirachta indica A. Juss. (Neem) from northern and western provenances of India. Plant Foods Hum Nutr. 1998;52:2938.

Krishna SH, Divakar S, Prapulla SG, Karanth NG. Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei. J Biotechnol. 2001;87:193201.

Lai CC, Zullaikah S, Vali SR, Ju Y. Lipase-catalyzed production of biodiesel from rice bran oil. J Chem Technol Biotechnol. 2005;80(3):3317.

Lakshminarayana G, Chandrasekhara Rao T, Ramalingaswamy PA. Varietal variations in content, characteristics and composition of mango seeds and fat. JOACS. 1983;60:889.

Lotero E, Liu Y, Lopez DE, Suwannakarn K, Bruce DA, Goodwin JG. Synthesis of biodiesel via acid catalysis. Ind Eng Chem Res. 2005;44:535363.

Ma F, Hanna MA. Biodiesel production: a review. Bioresour Technol.

1999;70:115.

Maranz S, Wiesman Z, Garti N. Phenolic constituents of shea (Vitellaria paradoxa) kernels. J Agric Food Chem. 2003;51:626873.

Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: a review. Renew Sustain Energy Rev. 2010;14:21732. Miller C, Austin H, Posorske L, Gonzlez J. Characteristics of an immobilized lipase for the commercial synthesis of esters. J AOCS. 1988;65:92731. Nelson L, Foglia T, Marmer W. Lipase-catalyzed production of biodiesel. JOACS.

1996;73:11915.

Pokoo-Aikins G, Nadim A, El-Halwagi M, Mahalec V. Design and analysis of biodiesel production from algae grown through carbon sequestration. Clean Technol and Env Policy. 2010;12:23954.

Sahoo PK, Das LM. Process optimization for biodiesel production from Jatropha, Karanja and Polanga oils. Fuel. 2009;88:158894.

Schenk P, Thomas-Hall S, Stephens E, Marx U, Mussgnug J, Posten C, Kruse O,

Hankamer B. Second generation biofuels: high-efficiency microalgae for biodiesel production. BioEnergy Res. 2008;1:2043.

Nde et al. AMB Expr (2015) 5:83

Page 12 of 12

Selmi B, Thomas D. Immobilized lipase-catalyzed ethanolysis of sunower oil in a solvent-free medium. JOACS. 1998;75:6915.

Shah S, Gupta MN. Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system. Proc Biochem. 2007;42:40914.

Shieh CJ, Liao HF, Lee CC. Optimization of lipase-catalyzed biodiesel by response surface methodology. Bioresour Technol. 2003;88:1036.

Sha S, Shweta S, Gupta MN. Enzymatic transesterication for biodiesel production. Indian J Biochem Biophys. 2003;40:3929.

Suganya T, Gandhi NN, Renganathan S. Production of algal biodiesel from marine macroalgae Enteromorpha compressa by two step process: optimization and kinetic study. Bioresour Technol. 2013;128:392400.

Suganya T, Kasirajan R, Renganathan S. Ultrasound-enhanced rapid in situ transesterication of marine macroalgae Enteromorpha compressa for biodiesel production. Bioresour Technol. 2014;156:28390.

Tan T, Lu J, Nie K, Deng L, Wang F. Biodiesel production with immobilized lipase: a review. Biotechnol Advances. 2010;28:62834.

Tiwari AK, Kumar A, Raheman H. Biodiesel production from jatropha oil

(Jatropha curcas) with high free fatty acids: an optimized process. Biomass Bioenergy. 2007;31:56975.

Tran D-T, Yeh K-L, Chen C-L, Chang J-S. Enzymatic transesterication of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour Technol. 2012;108:11927. Wang Y, Wu H, Zong MH. Improvement of biodiesel production by lipozyme

TL IM-catalyzed methanolysis using response surface methodology and acyl migration enhancer. Bioresour Technol. 2008;99:72327.

Yamane T. Enzyme technology for the lipids industry: an engineering overview. JOACS. 1987;64:165762.

Word count: 7806

Show less

The Author(s) 2015

Abstract

Translate

Mango, neem and shea kernels produce non-conventional oils whose potentials are not fully exploited. To give an added value to these oils, they were transesterified into biodiesel in a solvent-free system using immobilized enzyme lipozyme from Mucor miehei. The Doehlert experimental design was used to evaluate the methyl ester (ME) yields as influenced by enzyme concentration--EC, temperature--T, added water content--AWC, and reaction time--RT. Biodiesel yields were quantified by ^sup 1^H NMR spectroscopy and subsequently modeled by a second order polynomial equation with interactions. Lipozyme enzymes were more tolerant to high temperatures in neem and shea oils reaction media compared to that of mango oil. The optimum reaction conditions EC, T, AWC, and RT assuring near complete conversion were as follows: mango oil 7.25 %, 36.6 °C, 10.9 %, 36.4 h; neem oil EC = 7.19 %, T = 45.7 °C, AWC = 8.43 %, RT = 25.08 h; and shea oil EC = 4.43 %, T = 45.65 °C, AWC = 6.21 % and RT = 25.08 h. Validation experiments of these optimum conditions gave ME yields of 98.1 ± 1.0, 98.5 ± 1.6 and 99.3 ± 0.4 % for mango, neem and shea oils, respectively, which all met ASTM biodiesel standards.

Details

Title

ESolvent-free, enzyme-catalyzed biodiesel production from mango, neem, and shea oils via response surface methodology

Author

Nde, Divine Bup; Astete, Carlos; Boldor, Dorin

Pages

1-12

Publication year

2015

Publication date

Dec 2015

Publisher

Springer Nature B.V.

e-ISSN

21910855

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1186/s13568-015-0172-x

ProQuest document ID

1773207706

The Author(s) 2015

ESolvent-free, enzyme-catalyzed biodiesel production from mango, neem, and shea oils via response surface methodology

Jump to:

Full Text

Abstract

Details

Suggested sources