Optimization and kinetic studies on algal oil extraction from marine macroalgae Ulva lactuca

Bioresource Technology 107 (2012) 319–326

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Optimization and kinetic studies on algal oil extraction from marine macroalgae Ulva lactuca Tamilarasan Suganya, Sahadevan Renganathan ⇑ Department of Chemical Engineering, Alagappa College of Technology, Anna University, Chennai 600025, India

a r t i c l e

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Article history: Received 12 September 2011 Received in revised form 5 November 2011 Accepted 10 December 2011 Available online 19 December 2011 Keywords: Biodiesel Marine macro algae Ulva lactuca Oil extraction Extraction kinetics

a b s t r a c t In this present investigation, kinetic studies on oil extraction were performed in marine macroalgae Ulva lactuca. The algal biomass was characterized by scanning electron microscopy and Fourier TransformInfra Red Spectroscopy. Six different pre-treatment methods were carried out to evaluate the best method for maximum oil extraction. Optimization of extraction parameters were performed and high oil yield was obtained at 5% moisture content, 0.12 mm particle size, 500 rpm stirrer speed, 55 °C temperature, 140 min time and solvent-to-solid ratio as 6:1 with 1% diethyl-ether and 10% methylene chloride in n-hexane solvent mixture. After optimization, 10.88% (g/g) of oil extraction yield was achieved from 30 g of algal biomass. The rate constant was obtained for the first order kinetic study by differential method. The activation energy (Ea) was calculated as 63.031 kJ/mol. From the results obtained in the investigation, U. lactuca biomass was proved to be a suitable source for the biodiesel production. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Biodiesel seems to be a viable choice, but the most significant drawback is the cost of crop oils, that accounts for 80% of total operating cost, used for the biodiesel production (Demirbas, 2007). Biodiesel is usually produced from oleaginous crops such as rapeseed, soybean, sunflower and palm (Gouveia and Oliveira, 2009). Moreover, the availability of the oil crops serve as the sources for the biodiesel production are limited (Chisti, 2008). Therefore, it is necessary to find new feedstock suitable for biodiesel production, which does not drain on the edible vegetable oil supply. One alternative to the conventional oil crop is the algae because they contain oil, suitable for esterification/transesterification reaction for the biodiesel production. Biodiesel production from algae is widely considered as one of the most efficient methods. It appears to represent the recent renewable source of oil that could meet the global demand for transport fuels (Miao and Wu, 2006). The occurrence of algal blooms greatly disturbs the ecosystems by modifying food chains and faunal community structure. The accumulation of algal biomass relocating natural communities of sea grasses and higher plants (Taylor et al., 1995). Similarly, macroalgal blooms cause changes in the main biogeochemical cycles of C, N, P and S (Viaroli et al., 2001). As a result of this problem there is much attention needed to clean up the macro algae biomass. Hence this biomass is utilized for the production of biodiesel. ⇑ Corresponding author. Tel.: +91 9941613532; fax: +91 4422352642. E-mail address: [email protected] (S. Renganathan). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.045

Only less information is available for the production of biodiesel from marine macro algae and limited research work was observed for the extraction of oil from marine macro algae. Extraction is one of the fundamental processing steps used for recovering oil from biomass for the production of biodiesel. Various methods are available for the extraction of algal oil, such as mechanical, enzymatic, chemical extraction through different organic solvents and supercritical extraction. Solvent extraction is a common and an efficient technique for oil extraction. Solvent extraction involves the transfer of a soluble fraction from a solid material to a liquid solvent. Commercial grade n-hexane was used as a solvent for the extraction of oil from biomass for many years (Amin et al., 2010). Ulva lactuca is a thin flat green algae. The margin is ruffled and often torn. The membrane is thick, soft and translucent, and also grows without a stipe. This species in the Chlorophyta is formed of two layers of cells irregularly arranged. In this investigation, oil extraction from U. lactuca was studied. The main aim of the study was to find the efficiency of algal oil extraction using 6 different extraction methods and 12 different solvent systems. Optimization study for extraction was established with various parameters, such as moisture content, particle size, stirrer speed, extraction temperature, extraction time and solvent-to-solid ratio to obtain maximum oil extraction. The rate constant and activation energy was determined using the first order rate kinetics for the extraction of oil from U. lactuca biomass.

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2. Methods 2.1. Materials Organic solvents of analytical grade (Extra pure 99%) were purchased from Merck Ltd., Mumbai, India. They were reused after preliminary distillation. 2.2. Collection of algal sample U. lactuca belongs to green algae chlorophyta family. U. lactuca was collected from Rameswaram, Mandapam, South coast (Gulf of Mannar), India. The macroalgae was collected by hand picking from the intertidal and sub tidal regions. Sample collection was carried out during low tide period. 2.3. Preparation of U. lactuca algal biomass

of 15 lbs and time duration of 5 min (Kasai et al., 2003), (3) deep freezing pre-treatment was carried out using deep freezer. The algal biomass sample was placed under freezing conditions at 20 °C, (4) microwave pre-treatment was conducted in the microwave oven for 5 min time duration at 100 °C, 500 W and 2455 MHz (Lee et al., 2010), (5) lyophilization was carried out at 4 °C under vacuum pressure (14 Pa) using lyophilizer and (6) bead-beater pre-treatment was performed with 1 mm glass beads at high speed of 1500 rpm. After pre-treatment, the algal biomass was separated from water by using filtration technique through filter paper. Then the algal biomass was dried in hot air oven to maintain specific moisture content (Kabutey et al., 2011). The different moisture content % (MC%) of the algal biomass obtained were calculated from Eq. (1) (Kabutey et al., 2011):

MC ð%Þ ¼

Mi  Mf  100 Mi

ð1Þ

The collected algae was brought to laboratory and it was washed with fresh water followed by distilled water to separate potential contaminants such as adhering impurities, sand particles, epiphytes and animal castings. The samples were spread for shade drying. The dried biomass was grounded and particle size distribution was determined using a sieve analyzer as per ASTM standards.

where Mi is weight of the sample in the initial state (g) and Mf is weight of the sample after drying (g). The dried algal biomass with specific moisture content was allowed to mix with solvent mixture for the extraction of oil. After particular time of extraction, the slurry was transferred to a separating funnel to separate solvent–oil mixture and biomass. Solvent was separated from oil by distillation. All experiments were conducted with triplicate.

2.4. Characteristics of U. lactuca algal species

2.7. Solvent systems for oil extraction

To gain further insight into the effect of the ultra sonic pretreatment on the algal oil extraction, the microstructure of the algal biomass was analyzed with scanning electron microscopy (SEM, JEOL Ltd, Tokyo, Japan). The analysis was carried out for the algal biomass before and after treating with ultra sonication to identify the changes in the surface morphology. The presence of various functional groups in the algal biomass was analyzed using Fourier Transform Infra Red Spectroscopy (Spectrum RX1, US).

After the destruction of algal cells by ultrasonication, 12 different solvent systems were used for oil extraction such as n-hexane, methyl tertbutyl ether, chloroform:methanol (1:1), n-hexane:ether (3:1), chloroform:methanol (2:1), 1% diethyl ether and 10% methylene chloride in n-hexane, chloroform:2 propanol (2:1), hexane:2 isopropanol (3:2), dichloromethane:methanol (1:1), dichloromethane:ethanol (1:1), acetone:dichloromethane (1:1) and hexane:ethyl alcohol (1:1). During the selection of solvent study, the solvent-to-solid ratio was maintained as 5:1 for the oil extraction from algal biomass.

2.5. Sequence strategy for oil extraction from biomass 2.8. Extraction experimental set up The extraction of oil from U. lactuca marine macro algal biomass was performed based on the following sequence: (a) pre-treatment was performed to destruct the algal cells with various methods to increase the efficiency of the extraction, (b) after destruction of algal cells and cell wall, the algal biomass was mixed along with solvent mixture placed in a temperature controlled extraction unit with a magnetic stirrer for agitation, (c) solvent systems were selected for oil extraction to increase the efficiency of oil extraction, (d) optimization study was carried out with various extraction parameters to achieve high oil yield, (e) the extraction parameters were optimized for the maximum oil extraction yield from algal biomass, and (f) kinetic study was carried out for the algal oil extraction from U. lactuca marine macro algae to determine the order of the reaction, reaction rate constant and activation energy for oil extraction.

Extraction set up mainly consists of a three necked round bottom flask (250 ml). The large neck in the middle of the flask was connected to a reflux condenser, a thermometer was placed in one of the two side necks and the third neck was used for taking samples during the extraction process. The flask was submerged in a temperature controlled water bath with magnetic stirrer. 2.9. Optimization of extraction parameters to enhance the oil yield There are many factors influencing the oil extraction yield. The extraction parameters such as moisture content (2–6%), particle size (0.359–0.104 mm), stirrer speed (200–600 rpm), extraction temperature (35–65 °C), extraction time (20–160 min) and solvent-to-solid ratio (3:1–7:1) were optimized to increase the oil extraction yield.

2.6. Destruction of algal cells 2.10. Determination of oil extraction yield Dry algal biomass of 30 g along with water (water to biomass ratio as 3:1) was taken into a 250 ml of conical flask. In order to compare the oil extraction yield with direct extraction using solvent, the following methods for destruction of algal cells were tested: (1) ultra sonication using ultra sonic probe at 24 kHz with constant temperature (50 °C ± 1) for 5 min, (2) heat treatment was performed using auto clave. The experimental conditions for autoclave method were maintained as temperature of 121 °C, pressure

Oil extraction yield was calculated with respect to time for different temperatures. The oil extraction yield (% w/w) was calculated using the following Eq. (2) (Gutierrez et al., 2008):

Oil extraction yield ð%Þ ¼

Weight of oil extracted ðgÞ Weight of algal biomass ðgÞ  100

ð2Þ

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2.11. Characterization of U. lactuca algal oil

3.2. Effects of extraction methods

Fatty acid composition was analyzed by Gas Chromatography– Mass Spectrometry (GC-MS-QP 2010, Shimadzu) equipped with VF-5 ms capillary column (length – 30 mm, diameter – 0.25 mm, film thickness – 0.25 lm). The column temperature of each run was started at 70 °C for 2 min, then raised to 300 °C and maintained at 300 °C for 10 min. GC conditions were: column oven temperature – 70 °C, injector temperature – 260 °C, injection mode – split, split ratio – 10, flow control mode – linear velocity, column flow – 1.51 ml/min, carrier gas – helium 99.9995% purity. MS conditions were: ion source temp – 200 °C, interface temp – 240 °C, scan range – 40–1000 m/z, solvent cut time – 5 min, MS start time – 5 (min), MS end time – 35 (min) and ionization – EI (70 eV).

Oil extraction yield from U. lactuca biomass was evaluated by six different extraction methods (Fig. 1). The highest oil extraction yield of 8.25% (g/g) was achieved in ultra sonication method, which was adopted for further study. The extraction efficiency of this method was 2.25 times higher than that of direct extraction and 0.56 times higher than that of bead-beater of algal cells. The bead beater pre-treatment method has been widely used for lipid extraction from microalgae (Lee et al., 1998). An autoclave method is a heat treatment technique incorporated for oil extraction from the algal biomass. The oil extraction yield of 7.88% was obtained by this autoclave pre-treatment method. The membrane of U. lactuca is two cells thick. Hence, this heat treatment method is one of the very efficient method to destruct the membranes to enhance the oil yield. Deep-freezing, lyophilization and microwave pre-treatment methods caused partial hydrolysis and pre-esterification of the oil. These pre-treatment methods did not show much improvement in oil extraction yield when compared with direct extraction using solvent.

Molecular weight (g/mol) of algal oil can be calculated by using following Eq. (3) (Phan and Phan, 2008):

MW oil ¼ 3 

X ðMW i  % mi Þ þ 38

ð3Þ

where MWoil is average molecular weight of the oil (g), MWi is molecular weight of fatty acid i and % mi is percentage of fatty acid i. Chemical properties of algal oil were analyzed using titration methods. 3. Results and discussion 3.1. Characterization of U. lactuca algal biomass 3.1.1. Scanning electron microscopy analysis To further increase in the cell wall damage and porous surface of the algal cell, the ultra sonic pre-treatment method was implemented on the algal biomass for the oil extraction. Before and after the pre-treatment, the microstructure of the algal biomass was analyzed with scanning electron microscopy (SEM). From the SEM analysis, the surface morphology and porous surface of algal biomass which exposed for the solvent extraction was examined. It was also observed that the cell wall breakage caused by the ultrasonic cavitation energy and porous surface was found to be more on the surface of the biomass after making the pre-treatment when compared to the biomass without pre-treatment. This increase in porous surface leads to the increase in the oil extraction yield from the U. lactuca biomass due to the penetration of solvent to the inner surface of the algal biomass. This consequence is reflected in the fact that the oil extraction yield increased and obtained as 10.88% (g/g) from algal biomass with ultrasonic pretreatment. 3.1.2. Fourier Transform-Infra Red Spectroscopy analysis The FTIR spectra of the algal biomass showed the presence of the relevant functional groups. Alkynes bend (C–H bending) was observed at 669.87 cm1. The presence of hydroxyl group (O–H stretch) was characterized by the absorption peak at 3440.51 cm1. The ester group (C–O stretch or C–H bend) was identified at 1054.93 cm1. The presence of aromatic amine (C–N stretch) was confirmed by the absorption band at 1259.82 cm1. The peaks corresponding to the presence of alkenes (–C@C– stretch) were found at 1645.99 cm1. The alkane (C–H bending (scissoring)) group was identified at 1428.07. These peaks confirm the presence of triglyceride functional groups in the U. lactuca biomass. The presence of triglyceride groups for the different types of oils and fat were already reported by Yang et al. (2005). Similar type of result was previously reported by Patil et al. (2011) for the extraction of lipid from micro algae Nannochloropsis sps.

3.3. Ultra sonication pre-treatment Ultrasonication is an emerging powerful tool to accelerate many physical operations (Vilkhu et al., 2008). It has been also used to increase the oil extraction about three times of the conventional method used for the oil extraction. This ultra sonication pre-treatment method has many advantages over the other methods, such as reduced extraction time, reduced solvent consumption, greater penetration of solvent into cellular materials and improved release of cell contents into the bulk medium. This pre-treatment method may also provide more benefits economically and environmentally with health and safety aspects (Vilkhu et al., 2008). 3.3.1. Optimization of ultrasonic pre-treatment time Ultrasonic pre-treatment method improves extractions of oil significantly with higher efficiency, reduced extraction time and increased yield, as well as low moderate costs and negligible added toxicity. Algal biomass and water (water to biomass ratio as 3:1) were mixed in a flask. The ultra sonication was performed at 24 kHz with constant temperature (50 °C ± 1) and at different time intervals ranging from 2 to 6 min. It was observed that the increase in the ultra sonication pre-treatment time increased the oil yield from 2 to 5 min. At 5 min the yield was found to be 8.25% and the maximum oil extraction yield of 8.49% was obtained at 6 min duration of pre-treatment. After 6 min, the amount of oil yield

Oil extraction yield % (g/g)

2.12. Determination of molecular weight of algal oil

11 10 9 8 7 6 5 4 3 2 1 1

2

3

4

5

6

7

Pretreatment methods Fig. 1. Effects of various pre-treatment methods on oil extraction. 1. Direct extraction; 2. autoclave; 3. ultra sonication; 4. lyophilization; 5. bead-beater; 6. micro wave; 7. deep freezing.

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was found to be constant. From the literature, the benefit of using ultrasonic pre-treatment before extracting oil from the seeds of Jatropha curcas aqueous enzymatic oil extraction (AEOE) process was evaluated by Shah et al. (2005). Ultra sonic pre-treated J. curcas seeds provided significantly higher yield with reduction in extraction time. Thus, implementation of ultrasonic pre-treatment method reduced extraction time that may improve through put in commercial oil production process (Vilkhu et al., 2008). 3.4. Solvent system The selection of the solvent system for oil extraction from algal biomass is an important factor. Solvent selection for extraction of oil at the initial step would allow cost-effective for fuel production without further expense required for the purification of the product. The solvent chosen should have good extraction capacity and low viscosity to enhance the free circulation. An efficient extraction requires the penetration of solvent into the biomass and to match the polarity of the targeted compounds. An organic solvent has a higher solubility with oil, this solvent system was used to further degrade the cell walls of the algal biomass and to dissolve the oil to enhance the oil yield. The effect of solvent systems on algal oil extraction yield is shown in Table 1. 3.4.1. Hexane as a solvent system for extraction of oil Hexane is extensively used for oil extraction because of its high stability, low greasy residual effects, boiling point and low corrosiveness. The highest oil extraction yield of 8.75% was achieved from U. lactuca biomass using the most effective solvent mixture as 1% diethyl ether and 10% methylene chloride in n-hexane. This solvent system was used for the extraction of oil from the U. lactuca algal biomass for further studies. From the literature it was observed that the same solvent mixture was utilized for the extraction of triglycerides using the bond elut procedure (Kaluzny et al., 1985). Approximately 8.5% of oil extraction yield was achieved using n-hexane as a solvent for the algal oil extraction. Whereas the yield 8.16% was obtained using n-hexane–ether (1:1) as solvent mixture. While using n-hexane–2 isopropanol (Lee et al., 1998) and n-hexane–ethyl alcohol (Meziane et al., 2006) as solvent mixtures, the oil extraction yield was found to be 7.89% and 8.09%, respectively. Hexane is the most widely used solvent for the extraction of oil from micro and macro algae (Miao and Wu, 2006). Many research works were already established for the extraction of value added products from macro algal species with the use of hexane as extracting solvent (Aresta et al., 2003). Vijayaraghavan and Hemanathan (2009) used hexane as a solvent for oil recovery from fresh water algae for the biodiesel production. Large amount of microalgal oil was efficiently extracted from the heterotrophic cells by using hexane as a solvent (Miao and Wu, 2006).

3.4.2. Other solvent systems Oil extraction yield of 8.4% was achieved using single solvent methyl tert-butyl ether (MTBE). MTBE extraction allowed faster oil extraction and it forms the upper layer along with oil during phase separation, due to its low density. Approximately 7.12% and 7.36% of oil was extracted from algal biomass using chloroform and methanol as a solvent mixture using the method suggested by Bligh and Dyer (1959) and Folch et al. (1957). Chloroform–2 propanol mixtures achieved 7.32% of oil extraction yield. The same solvent system was used to separate all neutral lipids from crude mixture by Kaluzny et al. (1985). Nearly 7.51% and 7% of oil extraction yield was obtained using dichloromethane–methanol and dichloroethane–ethanol as solvent system, respectively. Lee et al. (1998) conducted an experiment with these solvent systems and extracted 18% of oil from Botryococcus braunii. Oil yield of 6.55% was obtained using acetone–dichloromethane as a solvent mixture. This solvent mixture extracted low oil yield from algal biomass. So this solvent mixture is not used for the further extraction experiment. 3.5. Optimization of various parameters influencing the extraction of oil The extraction of oil depends on the nature of the solvent, moisture content, particle size, stirrer speed, extraction temperature, time of extraction and solvent-to-solid ratio (Chaiklahan et al., 2008; Siddiquee and Rohan, 2011). 3.5.1. Effect of moisture content In algal oil extraction, moisture content is to be considered as an important factor. Fig. 2a shows the effect of moisture content on the oil extraction. Moisture content varying from 2% to 6% was used for the oil extraction from U. lactuca algal biomass. Effect of the moisture content was studied on oil extraction by keeping other parameters as constant (particle size – 0.161 mm diameter, stirrer speed 400 rpm, extraction temperature 50 °C, solvent-tosolid ratio 1:5 and extraction time 120 min). From the Fig. 2a, it was observed that the oil extraction was found to be increased with increase in moisture content up to 5%. The maximum oil extraction yield of 8.86% was achieved at 5% moisture content of biomass. Above this 5% moisture content, the oil extraction yield was found to be decreased with increase in moisture content. This may be attributed due to the presence of high level moisture content as a barrier between the solvent and algal biomass which may restrict the penetration of solvent into the biomass. Rao and Arnold (1958) studied the extraction of oil from oilseeds with the moisture level of 3% and maximum yield was obtained at this 3% moisture level.

Table 1 Effects of various solvent systems on oil extraction yield (%). S. No.

Solvent/solvent mixture

Ratio/percentage

Oil extraction yield (%)

1 2 3 4 5 6 7 8 9 10 11 12

Hexane Methyl tertbutyl ether Chloroform:methanol Hexane:ether Chloroform:methanol Diethyl ether and methylene chloride in hexane Chloroform:2 propanol Hexane:2 isopropanol Dichloromethane:methanol Dichloromethane:ethanol Acetone:dichloromethane Hexane:ethyl alcohol

– – 1:2 3:1 2:1 1% and 10% 2:1 3:2 1:1 1:1 1:1 1:1

8.53 ± 0.04 8.42 ± 0.09 7.36 ± 0.08 8.16 ± 0.10 7.12 ± 0.07 8.75 ± 0.09 7.32 ± 0.08 7.89 ± 0.06 7.51 ± 0.04 7.00 ± 0.05 6.55 ± 0.04 8.09 ± 0.03

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9 8.5 8 7.5 7 0

1

2

3

4

5

6

(b)

9 8 7 6 5 4 3

2 0.05

7

Oil extraction yield % (g/g)

(a)

Oil extraction yield % (g/g)

Oil extraction yield % (g/g)

10 9.5

0.1

0.15

Moisture content(%) 12

(d)

10 9 8 7 6 5 30

0.3

0.35

0.4

11

12

(e)

10 9 8 7 6 5 4

35

40

45

50

55

Temperature (°C)

200

300

60

65

70

0

20

40

60

80

100

Time (mins)

120

400

500

600

700

Stirrer Speed (rpm)

Oil extraction yield % (g/g)

11

0.25

(c)

Particle size (mm)

Oil extraction yield % (g/g)

Oil extraction yield % (g/g)

12

0.2

10 9.5 9 8.5 8 7.5 7 6.5 6 5.5 5 100

140

160

180

(f)

11.5 11 10.5 10 9.5 9 8.5 8 1

2

3

4

5

6

7

8

Solvent to Solid ratio

Fig. 2. Optimization of various parameters on oil extraction from U. lactuca biomass. (a) Effect of moisture content on oil extraction [conditions: particle size: 0.161 mm diameter; stirrer speed: 400 rpm; temperature: 50 °C; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (b) Effect of particle size on oil extraction [conditions: moisture content: 5%; stirrer speed: 400 rpm; temperature: 50 °C; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (c) Effect of stirrer speed on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; temperature: 50 °C; extraction time: 120 min, solvent-to-solid ratio:5:1]. (d) Effect of extraction temperature on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm; extraction time: 120 min; solvent-to-solid ratio: 5:1]. (e) Effect of extraction time on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm, temperature: 55 °C; solvent-to-solid ratio: 5:1]. (f) Effect of solvent-to-solid ratio on oil extraction [conditions: moisture content: 5%; particle size: 0.12 mm diameter; stirrer speed: 500 rpm; temperature: 55 °C; extraction time: 140 min].

3.5.2. Effect of particle size on oil extraction Particle size is found to be a critical parameter for the extraction of oil from the biomass. Smaller the size of biomass leads to greater in the interfacial area between the solid and liquid. Therefore the increase in interfacial area increases the oil extraction yield. The maximum oil extraction of 9.05% was obtained at 0.12 mm diameter of particle size with 5% optimum moisture level at 50 °C temperature with 400 rpm and solvent-to-solid ratio of 5:1 for 120 min. From the Fig. 2b, it was observed that the oil extraction yield was found to be gradually increased from 6.57% to 9.05% with decrease in particle size from 0.359 mm to 0.12 mm diameter. Below 0.12 mm diameter of the particle, there was no any further increase in oil extraction. This shows that the maximum oil extraction yield of 9.05% was obtained with the biomass particle size of 0.12 mm diameter. It is well recognized that the rate of solvent extraction is controlled by the biomass particle size. The lower extraction rate is attributed due to the bigger particles. This bigger particle creates difficulty for the solvent to penetrate into the core of the biomass to leach the oil. It is noticed that the particle size is not only increases the extraction rate, but also increases the oil extraction yield. Qian et al. (2008) showed that the extraction rate of cottonseed oil was found to increase with the reduced particle size of cottonseed flours. However, further decrease in the particle size did not show much improvement in the oil extraction yield of cottonseed oil. Particle size varying from 0.3 mm to 0.335 mm was found to be optimum for cottonseed flour. According to Han et al. (2009), the main reason for increasing oil yield was due to decrease in particle size, which in turn increases the specific surface area of oilseed interacting with the solvent. 3.5.3. Effect of stirrer speed on oil extraction The effect of stirrer speed on oil extraction is illustrated in Fig. 2c. Stirrer speed of the extraction also affects the oil yield. It increases the eddy diffusion and the transfer of oil from the slurry form of the algal biomass to the solvent mixture. The effect of stirrer speed on oil extraction in the range of 200–600 rpm was evaluated with other parameters as constant. The oil extraction yield

was found to be increased from 7.81% to 9.36% with an increase in the stirrer speed. The maximum oil extraction yield was obtained as 9.36%, at 500 rpm. However, for stirrer speed more than 500 rpm, there was no significant increase in the oil extraction yield from U. lactuca biomass in slurry. From the experimental result, it was observed that oil extraction yield was found to be low at low stirrer speed. In order to overcome this problem, the extraction was carried out at higher stirrer speed of 500 rpm. This is clearly revealed that the mass transfer plays major role during extraction with solvent system. Kadi and Fellag (2001) studied the effect of stirrer speed on oil extraction from olive foot cake using hexane as a solvent. They have extracted oil from 6.9% to 7.7% with the use of stirrer speed varying from 600 to 1000 rpm. Maximum yield of 7.7% was achieved at 800 rpm. 3.5.4. Effect of temperature The effect of temperature on the algal oil extraction yield was examined over the range of 35–65 °C from U. lactuca biomass (Fig. 2d). The oil yield was found to be enhanced with the rise in the temperature. This is due to the increase in the dissolution capacity of the solvent system. The rise in the temperature from 35 to 55 °C leads to increase in the yield from 7% to 9.75%. At 55 °C, highest oil extraction yield of 9.75% was obtained at optimum conditions of 5% moisture content, 0.12 mm particle size and 500 rpm stirrer speed for 120 min. Solvent-to-solid ratio of 5:1 was maintained. It was observed that at optimum temperature of 55 °C, the solubility of the solvent was found to be increased with increase in diffusion rate (Denery et al., 2004). Solvent based extraction was developed because it allows complete extraction of oil in the low temperature varying from 50 to 60 °C (Amin et al., 2010). Karlovic et al. (1992) investigated the effect of temperature on the kinetics of oil extraction from corn germ flakes prepared by a dry degermination process. Increase in the temperature can enhance the capacity of solvents to dissolve the oil because the thermal energy can overcome the cohesive and adhesive interactions (Karlovic et al., 1992).

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3.5.5. Effect of extraction time The extraction time is an important parameter for oil yield. It helps in deciding the optimum residence time required for the extraction process. In this study, the effect of time on the oil extraction was investigated with different time intervals varying from 20 to 160 min (Fig. 2e). The results showed that oil extraction yield increases with increase in time. The extraction was established with the optimum condition of 5% moisture content, 0.12 mm diameter of particle size, stirrer speed of 500 rpm, temperature at 55 °C and solvent-to-solid ratio of 5:1. After 120 min of extraction, the oil extraction yield was obtained as 9.75%. Although the time was extended up to 140 min, the oil extraction yield was found to be improved up to 10.59%. The increase in extraction time above 140 min did not show any further significant improvement in the extraction. Hence, 140 min was found to be an optimum extraction time for further studies. The oil extraction yield was studied at different time intervals at constant temperature. The oil extraction yield was gradually increased from 4.1% to 7.89% with increase in time from 20 to 160 min at constant temperature of 35 °C. The same type of study was conducted for different temperatures varying from 35 to 55 °C. The oil extraction yield was found to be maximum at 55° C when compared to all other temperatures studied for extraction with the time duration of 140 min. From the above data the kinetic model was analyzed for the oil extraction. Higher oil extraction yield was obtained with much shorter time (140 min) than with other extraction methods such as aqueous extraction and enzyme assisted aqueous extraction (18–25 h). This short extraction time could be due to the rapid mass transfer of oil. From the literature, it was observed that the extraction of oil from Cyanobacterium spirulina was carried out at different time interval varying from 50 to 150 min. The optimum time was found to be 120 min for the maximum production of oil (Chaiklahan et al., 2008). The maximum yield of 26.4% oil from Cunninghamella echinulata CCF-103 fungus species at the time duration of 180 min was reported by Certik and Horenitzky (1999). 3.5.6. Effect of solvent-to-solid ratio The effect of solvent-to-solid ratio on the oil extraction is shown in Fig. 2f. The influence of solvent-to-solid ratio from 3:1 to 7:1 on oil extraction was studied by maintaining all other parameters at optimum conditions. As the solvent-to-solid ratio increased from 3:1 to 6:1, the oil yield was found to be increased from 9% to 10.88%. The trend was continued with increase in solvent-to-solid ratio up to 6:1. Further increase in solvent-to-solid ratio above 6:1 did not show much improvement in the oil extraction. Therefore the solvent-to-solid ratio of 6:1 (v/w) was found to be an optimum ratio for the further study. Kadi and Fellag (2001) used solvent-to-solid ratio as 4:1 for the extraction of oil from olive foot cake using hexane as a solvent and obtained 9.4% yield. Pokoo-Aikins et al. (2010) extracted oil from sewage sludge by using toluene, n-hexane, ethanol and methanol as a solvent mixture. They used solvent to sludge ratio as 5:1 for the maximum extraction of oil. The oil extraction yield as 10.88% obtained from the present investigation stands in comparison to other established experimental data. Hossain and Salleh (2008) extracted algal oil from Oedogonium sp. (fresh water macro algae) for the biodiesel production. They extracted 3 g of oil from 32.4 g of the algal biomass. The maximum oil extraction yield was found to be 9.2% from Oedogonium macroalgae biomass (Hossain and Salleh, 2008). 3.6. Composition analysis and properties of algal oil Fatty acid composition of algal oil was analyzed by GC–MS (Table 2). From the analysis, the saturated fatty acids (79.82%)

were found to be more when compared to unsaturated fatty acids (20.18%). In the saturated fatty acid Palmitic acid composition was observed to be maximum of 50.16% in U. lactuca algal oil. Properties such as molecular weight, Saponification value, Iodine value, Acid value and free fatty acid (FFA) were determined (Table 3). 3.7. Extraction kinetics A reaction rate equation for oil extraction from algal biomass can be written as Eq. (4) (Topallar and Gecgel, 2000)

dY=dt ¼ kY

n

ð4Þ

where Y is the oil extraction yield (%), t is the time of extraction (min), k is the extraction rate constant (min1) and n is the order of the reaction. As the percentage of oil extraction increased in the course of time (Table 4), the terms dY/dt have a positive sign (Topallar and Gecgel, 2000). Using the values in Table 4 and applying the differential method, plots of ln Y versus ln (dY/dt) at different temperatures with optimum conditions were found to be linear according to Eq. (4). A first-order kinetic model was fitted well with average regression coefficient (R2) value obtained as 0.936. The reaction rate constants and the order of the reaction were determined using the intercept and slope of the liner plot (Fig. 3). From the analysis of the data, the oil yield was found to be increased with increase in extraction time. The yield was also analyzed with respect to the extraction time at constant temperature ranging from 35 to 55 °C. The determination of reaction rate constant explains about the time required to get maximum extraction of oil from the U. lactuca biomass. Rate constants determined from the plots were found to be increased with increase in temperature. This may be due to the increase in the reactivity of the solvent to enhance the rate of extraction. Hence, reaction rates are often found to depend strongly on temperature.

Table 2 Fatty acid profile of U. lactuca algal oil. Name of the fatty acid

No. of carbon atoms

Relative %

Lauric acid Tridecanoic acid Myristic acid Pentadecanoic acid Palmitic acid Heptadecanoic acid Streacic acid Nonadecanoic acid Arachidic acid Heneicosanoic acid Behinic acid Oleic acid Linoleic acid Linolenic acid

12:0 13:0 14:0 15:0 16:0 17:0 18:0 19:0 20:0 21:0 22:0 18:1 18:2 18:3

3.08 3.72 5.85 1.77 50.16 0.75 11.07 0.02 2.05 0.23 1.12 16.57 3.23 0.38

Table 3 Properties of algal oil extracted from U. lactuca biomass. Parameters

Extracted algal oil

Units

Average molecular weight Saponification value Iodine value Acid value FFA

780 212.9 ± 1.86 97.22 ± 1.33 14.27 ± 1.12 6.6 ± 0.35

g/mol mg of KOH/g – mg of KOH/g wt%

325

T. Suganya, S. Renganathan / Bioresource Technology 107 (2012) 319–326 Table 4 The oil extraction yield % from algal biomass at various extraction temperatures with respect to extraction time. Temp (°C)

Reaction rate constants (k) (min1)

35 40 45 50 55

5.27  104 2.33  103 2.88  103 5.53  103 8.14  103

Oil extraction yield (%) 20 min

40 min

60 min

80 min

100 min

120 min

140 min

160 min

4.15 ± 0.08 4.25 ± 0.09 4.48 ± 0.07 4.68 ± 0.05 5.31 ± 0.07

4.80 ± 0.09 4.80 ± 0.10 4.95 ± 0.08 5.30 ± 0.09 6.68 ± 0.08

5.13 ± 0.11 5.36 ± 0.10 5.60 ± 0.09 6.11 ± 0.07 7.36 ± 0.09

5.60 ± 0.10 6.10 ± 0.11 6.38 ± 0.09 7.05 ± 0.12 8.08 ± 0.11

6.30 ± 0.09 6.90 ± 0.12 7.41 ± 0.08 8.16 ± 0.11 8.88 ± 0.12

7.00 ± 0.08 8.12 ± 0.11 8.59 ± 0.10 9.36 ± 0.11 9.75 ± 0.12

7.80 ± 0.07 8.88 ± 0.09 9.79 ± 0.09 10.26 ± 0.09 10.59 ± 0.09

7.89 ± 0.06 8.90 ± 0.08 9.60 ± 0.08 10.20 ± 0.07 10.59 ± 0.09

ln Y

-1.0 1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5

-1.4 T = 308 K y = 1.521x -6.261 R² = 0.904 -1.8

T = 318 K y = 1.523x -6.063 R² = 0.937 T = 313 K y = 1.384x -5.851 R² = 0.933

-2.2

T = 323 K y = 1.161x -5.197 R² = 0.948 T = 328 K y = 0.717x -4.811 R² = 0.956

ln (dY/dt)

-2.6 -3.0 -3.4 -3.8 -4.2 -4.6 -5.0

Fig. 3. A plot of [ln (dY/dt)] versus [ln Y] at different temperatures ranged from 35 to 55 °C for extraction of oil from U. lactuca biomass.

1 / T (K-1) -3 0.003 -3.5 -4

0.00305

0.0031

0.00315

0.0032

0.00325

y = -7581.x + 18.21 R² = 0.943

-4.5

ln K

-5 -5.5 -6 -6.5 -7 -7.5 -8 Fig. 4. Activation energy calculation from the plot of ln k versus 1/T (K1).

0.0033

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3.8. Calculation of activation energy The relation between rate constant versus extraction temperature can be described by the Arrhenius Equation (Eq. (5)) (Topallar and Gecgel, 2000):

k ¼ AeEa=RT

ð5Þ 1

where k is the reaction rate constant (min ), A is the Arrhenius constant or frequency factor (s1), Ea is the activation energy (kJ/ mol), R is the universal gas constant (J/mol K) and T is the absolute temperature in K. A plot of ln k versus 1/T gives a straight line whose slope represents the activation energy of extraction – Ea/R (Fig. 4) (Topallar and Gecgel, 2000). Thus, the activation energy (Ea) was calculated as 63.031 kJ/mol. The same type of result was previously reported by Amin et al. (2010) for the extraction oil from J. curcas. 4. Conclusion Extraction of oil from U. lactuca algal biomass using ultrasound pre-treatment method showed better results when compared with other methods studied. Diethyl ether of 1% and 10% of methylene chloride in n-hexane achieved high oil yield. The maximum oil yield of 10.88% was obtained with optimum conditions of 5% moisture content, 0.12 mm of particle size, 55 °C temperature, 500 rpm stirrer speed, solvent-to-solid ratio as 6:1 and 140 min extraction time. Kinetic studies revealed that this extraction followed first order. The oil extracted from U. lactuca was found to be one among the suitable source for biodiesel production. Acknowledgements The authors gratefully acknowledge Department of Science and Technology (DST), New Delhi for providing financial support to carry out this research work under PURSE scheme. One of the authors Ms. T. Suganya is grateful to DST, New Delhi for the award of DSTPURSE fellowship. References Amin, S.K., Hawash, S., El Diwani, G., El Rafei, S., 2010. Kinetics and thermodynamics of oil extraction from Jatropha curcas in aqueous acidic hexane solutions. J. Am. Sci. 6, 293–300. Aresta, M., Dibenedetto, A., Tommasi, I., 2003. Energy from macro-algae. Fuel Chem. 48, 26. Bligh, E.C., Dyer, W.I., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917. Certik, M., Horenitzky, R., 1999. Supercritical CO2 extraction of fungal oil containing Linolenic acid. Biotechnol. Tech. 13, 11–15. Chaiklahan, R., Chirasuwan, N., Loha, V., Bunnag, B., 2008. Lipid and fatty acids extraction from the Cyanobacterium spirulina. Sci. Asia 34, 299–300. Chisti, Y., 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26, 126–131. Demirbas, A., 2007. Importance of biodiesel as transportation fuel. Energy Policy 35, 4661–4670. Denery, J.R., Dragull, K., Tang, C.S., Li, Q.X., 2004. Pressurized fluid extraction of carotenoids from Haematococcus pluvialis and Dunaliella salina and kavalactones from Piper methysticum. Anal. Chim. Acta 501, 175–181.

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