In situ ethyl ester production from wet algal biomass under microwave-mediated supercritical ethanol conditions

Bioresource Technology 139 (2013) 308–315

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In situ ethyl ester production from wet algal biomass under microwave-mediated supercritical ethanol conditions Prafulla D. Patil a,1, Harvind Reddy a, Tapaswy Muppaneni a, Tanner Schaub b, F. Omar Holguin b, Peter Cooke c, Peter Lammers d, Nagamany Nirmalakhandan e, Yin Li a,f, Xiuyang Lu f, Shuguang Deng a,f,⇑ a

Chemical Engineering Department, New Mexico State University, Las Cruces, NM 88003, USA Chemical Analysis and Instrumentation Laboratory, New Mexico State University, Las Cruces, NM 88003, USA Electron Microscopy Laboratory, New Mexico State University, Las Cruces, NM 88003, USA d Energy Research Laboratory, New Mexico State University, Las Cruces, NM 88003, USA e Civil and Environmental Engineering Department, New Mexico State University, Las Cruces, NM 88003, USA f Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Direct conversion of wet algae to

biodiesel under supercritical ethanol conditions.  Study the effects of algae to ethanol ratio and reaction time on algal biodiesel yield.  Controlled microwave power conditions with passive heating elements (SiC).  No expensive and extensive harvesting and drying steps for algal biodiesel production.  In situ transesterification using a green solvent and catalyst-free approach.

a r t i c l e

i n f o

Article history: Received 24 February 2013 Received in revised form 9 April 2013 Accepted 11 April 2013 Available online 20 April 2013 Keywords: Microwave irradiation Supercritical ethanolysis Algal biomass Biodiesel In situ transesterification

a b s t r a c t An in situ transesterification approach was demonstrated for converting lipid-rich wet algae (Nannochloropsis salina) into fatty acid ethyl esters (FAEE) under microwave-mediated supercritical ethanol conditions, while preserving the nutrients and other valuable components in the algae. This single-step process can simultaneously and effectively extract the lipids from wet algae and transesterify them into crude biodiesel. Experimental runs were designed to optimize the process parameters and to evaluate their effects on algal biodiesel yield. The algal biomass characterization and algal biodiesel analysis were carried out by using various analytical instruments such as FTIR, SEM-EDS, TLC, GC–MS and transmission electron microscopy (TEM). The thermogravimetric analysis (TGA) under nitrogen and oxygen environments was also performed to examine the thermal and oxidative stability of ethyl esters produced from wet algae. This simple in situ transesterification process using a green solvent and catalyst-free approach can be a potentially efficient route for algal biodiesel production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction ⇑ Corresponding author at: Chemical Engineering Department, New Mexico State University, Las Cruces, NM 88003, USA. Tel.: +1 575 646 4346; fax: +1 575 646 7706. E-mail address: [email protected] (S. Deng). 1 Current address: American Refining Group, Inc. Bradford, PA 16701. 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.045

Global demand for energy has been projected to increase by 49% by the year 2035 (EIA, 2012). As energy demand around the world increases, there is an increasingly intense competition for

P.D. Patil et al. / Bioresource Technology 139 (2013) 308–315

diminishing petroleum reserves. The current fossil fuel-based economy is unsustainable due to the negative environmental impacts, economic dependence, and energy security issues, and there is an urgent need to find a sustainable solution for this serious energy crisis (Demirbas, 2009). Biomass is one of the most suitable renewable energy sources, provided there are developments in effective new technologies for the generation of this bio-energy. This approach offers a great potential for the long-term storage of carbon and would contribute to a closed carbon cycle as well as reduce the greenhouse gas emissions compared with fossil fuels. The efficient utilization of biomass is an extremely important area for sustainable energy development (Klass, 1998). Many pathways are currently under consideration for producing liquid fuels from biomass to supplement the fossil-based liquid transportation fuels. The most promising among these are algaebased advanced biofuels as high energy density fungible fuels for aviation, nautic and ground transport. Culturing microalgae as an alternative feedstock for biodiesel has drawn a special attention for many reasons including fast growth rate, potential for high lipid content, ability to capture carbon dioxide (CO2) emissions, utilizing a wide variety of water sources, compatible with integrated production of fuels and co-products within biorefineries, removing pollutants from wastewater streams, effective land utilization and eliminating the dilemma in the food versus fuel debate (Hossain and Salleh, 2008; Meng et al., 2009). Three major components can be extracted from algal biomass: lipids (including triglycerides and fatty acids), carbohydrates, and proteins. While lipids and carbohydrates are fuel precursors (e.g. gasoline, biodiesel, ethanol, and jet fuel), proteins can be used for co-products (e.g. animal/fish feeds). An ideal algal biodiesel production process consist of a few key steps including algae cultivation, algae harvesting, lipid extraction, lipid to biodiesel fuel conversion and utilization of co-products from extracted algal oils and lipid extracted algae (Chisti, 2007). The extensive upstream and downstream processing makes algal biodiesel production an expensive and tedious process. In addition, there are certain techno-economical challenges in extractive-conversion process that prevent the successful commercialization of algal biodiesel. The major challenges are: (a) lack of effective and efficient harvesting technique; (b) high energy consumption in algae drying; (c) consumption of large amounts of hazardous chemicals in lipid extraction; (d) excessive lipid separation and purification; (e) expensive conversion processes. Therefore, it is critical to develop an easy and environmentally benign conversion or one-step method that reduces the chemical and energy consumption, and duration of the overall algal biodiesel production process. There are a few potential studies that have reported the direct conversion of algal biomass to alkyl esters. These investigations using a one-pot approach are: (1) biodiesel production from wet algal biomass through in situ lipid hydrolysis and supercritical transesterification (Levine et al., 2010); (2) direct transesterification of a microalgae biomass using conventional heating with sulfuric acid as a catalyst (Johnson and Wen, 2009); (3) direct conversion (a one-stage method) of dry algal biomass to biodiesel production using microwave and ultrasound radiation with the aid of a SrO catalyst (Koberg et al., 2011); (4) alkali-catalyzed in situ transesterification of Spirulina sp. by methanol in reactions containing various solvents (Xu and Mi, 2011); (5) producing fatty acid alkyl esters directly from a biomass using an alkylation reagent (Hatcher and Liu, 2009). All these conversion steps including extraction, transesterification, and purification are all energy-and cost-intensive and strictly limited by nature of feedstock input, purity of reaction product, nutritional value of lipid extracted algae (LEA) and scale-up difficulties. Other significant problems in the direct algal biodiesel conversion process are long reaction time, controlling the process parameters such as temperature and pressure,

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processing feedstocks with high free fatty acid (FFA) contents, use of toxic solvents and optimization of process parameters for maximum yield. Herein, we report a novel integrated approach for direct conversion of wet algal biomass to biodiesel under microwave-mediated supercritical ethanol conditions. This process promotes a faster reaction mechanism, reduces energy consumption and extractive-transesterification time, and produces a significant yield of biodiesel. In addition, the application of catalyst-free synthesis with a green solvent, chemically safer reaction using microwaves and less contaminated by-product formation makes this biodiesel process ‘eco-friendly’ sustainable. The unique advantage of this process is that it also avoids expensive and extensive harvesting and drying steps for the production of biodiesel. Microwave irradiation has been used to extract oils from biomass, soils and vegetable feedstock (Kiss et al., 2000; Pan et al., 2002). The main advantage of using microwave accelerated organic synthesis is the shorter reaction time due to rate enhancement. The rate of reaction can be described by the Arrhenius equation as: K ¼ AeDG=RT , where ‘A’ is a pre-exponential factor, ‘DG’ is free energy of activation. The rate of chemical reaction can be increased through the pre-exponential factor A, which is the molecular mobility that depends on the frequency of the vibrations of the molecules at the reaction interface or the pre-exponential factor can be altered by affecting the free energy of activation (Lidstrom et al., 2001). Microwave-assisted heating is substantially more rapid than conventional heating as heat is delivered via radiation rather than convection and conduction. In this report, several experiments were carried out to identify and optimize the effect of the process parameters involved in the extraction and transesterification of algal biomass under microwave-mediated supercritical ethanol condition. When heated up to the near- or supercritical stage, organic solvents become more and more transparent to microwave irradiation and the energy transfer is less effective (Kremsner and Kappe, 2005). Therefore, we depict the use of a passive heating element made of silicon carbide (SiC) to augment the microwave-mediated heating process at high temperatures. The supercritical conditions for ethanol are above 63 bar and 241 °C, which can be reached easily under microwave irradiation. Analytical results for algal biodiesel obtained from gas chromatography–mass spectrometry (GC–MS), thermogravimetric analysis (TGA), thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR) are also presented. 2. Methods Wet algal paste (Nannochloropsis salina sp.) provided by the Solix Biofuels Inc was used in this study. For GC–MS analysis, C4–C24 even carbon saturated FAEEs mixture (1000 mg/mL in hexane, 49454U) and ethyl heptadecanoate, internal standard were purchased from SupelcoÒ. Extra pure ethanol, hexane, sodium sulfate and activated carbon were procured from Acros Organics. For TLC analysis, acetic acid, diethyl ether, sodium sulfate and sulfuric acid were produced from Acros Organics, NJ, USA. All chemicals were at least of A.C.S. grade or better. The experiments were performed in an Anton Paar Synthos 3000 microwave reactor, utilizing four vessels in the Rotor 8SXQ80 (80 mL quartz vessels, 0–80 bar, 25– 300 °C, 0–1400 W) with silicon carbide (SiC) as a passive heating element. 2.1. Characteristics of N. salina algal cells Qualitative elemental analysis of algal biomass was determined by scanning electron microscopy (model S-3400N, Hitachi High Technologies, Pleasanton, CA, USA) equipped with an energy-dispersive X-ray spectrometer (Noran System Six 300, Thermo

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Electron Corp., Madison, WI, USA). The major elements and their approximate composition in (wt.%) were carbon (74.96%), oxygen (20.34%), sodium (0.99%), magnesium (0.37%), silicon (0.19%), phosphorous (0.49%), chlorine (1.08%), and potassium (0.85%), calcium (0.39%), sulfur (0.33%). The total lipid content in algal biomass was determined as 50–55% (% of dry biomass) by the Folch method. The lipid composition of the algal samples indicates the presence of maximum amount of triglyceride (TAG) percentage (around 70–75%) followed by polar lipids and hydrocarbon percentage. From thermogravimetric analysis (TGA) differential plot for wet algae, the first region of weight loss occurred at about 100–150 °C due to dehydration of biomass. The percent loss of water occurred in the first region was about 60% of the initial weight of the sample and represents the moisture content in the wet algal biomass.

2.2. Experimental procedure The experimental protocol for the single-step microwave-mediated supercritical ethanol extraction and transesterification process for algal (N. salina) biomass is illustrated in Fig. 1. The reactions were conducted using wet algal biomass (2 g) under a set of conditions: reaction times (hold time) of 10–30 min; algae to ethanol (wt./vol.) ratios of 1:6–1:15;controlled power dissipation of 1400 W and reduced to 800 W. Maximum power is only needed to heat the mixtures up to the near- or supercritical stage. Once the limit is reached, minimum power is needed to maintain these extreme conditions for several minutes. This ensures reduced material stress on vessels, sensors and rotor components and a prolonged lifetime of the accessories. Due to the use of the special holder the T-probe for accurate temperature measurement is not applied. The reaction is performed with a power-controlled run at a 280 °C IR limit. Actual inside temperature is approx. 35– 40 °C above sensed IR vessel surface temperature. Averages of test results obtained for four identical samples (utilized four vessels per run) maintained at same conditions were analyzed to evaluate the reproducibility of the microwave effect. The downstream process includes volatilization of ethanol, purification and separation of crude algal biodiesel (FAEEs) is described elsewhere (Patil et al., 2012). The solvent, n-hexane, was added to the remaining product resulted from vacuum distillation to induce biphasic layer and to extract non-polar lipids in

the upper organic phase during the centrifugation. The resulting product was dried with sodium sulfate and analyzed with GC– MS, FTIR, TGA and TLC. The by-product contaminants such as glycerol, traces of alcohol and other impurities remained in the lower aqueous phase. The algal cell-debris from the reaction mixtures was removed in the centrifugation and filtration step. Sulfur, nitrogen and phosphorous-containing compounds present in the algal lipid (mainly in the phospholipids and glycolipids) are likely transferred into the water-soluble fractions following the transesterification reaction. In addition, some sugars, acids and highly oxygenated compounds which would be soluble in the aqueous phase are possibly separated from organic fractions. The power method used for microwave transesterification process is described in Table 1 includes information about the hold time, ramp time, fan levels for heating and cooling of the reaction. 2.3. Microwave-mediated supercritical transesterification mechanism The microwave-mediated supercritical ethanol transesterification mechanism of algal biomass to yield ethyl ester is illustrated in Fig. 2. The supercritical state for the reaction mixture is induced by the microwave irradiation effect. The microwave effect on the transesterification reaction is twofold: (1) enhancement of reaction by a thermal effect, and (2) evaporation of ethanol due to the strong microwave interaction of the material (Loupy et al., 1993). Microwave thermal effect offers major benefits such as non-contact heating, rapid energy transfer (penetrative radiation), volumetric heating, reduction in thermal gradients, fast start-up and stooping, and reverse thermal effect. The microwave interaction with the reaction compounds (triglycerides and ethanol) results in a large reduction of activation energy due to increased dipolar polarization, ionic conduction, and interfacial polarization mechanisms. 2.4. Analytical methods The algal biodiesel samples were analyzed by a gas chromatography–mass spectrometry (GC–MS) system incorporated with an Agilent 5975C MSD (Triple-Axis Detector) and an Agilent 7890 A GC equipped with a capillary column (DB-23, 60 m  250 lm  0.15 lm nominal). FAEE yield is expressed as a percentage of dry algal weight (i.e. g FAEE extracted/g dried algae). The

Fig. 1. Experimental protocol for microwave-mediated supercritical ethanol transesterification process for algal biomass.

P.D. Patil et al. / Bioresource Technology 139 (2013) 308–315 Table 1 Power method used for microwave-mediated SCE transesterification process. Step

Power (W)

Ramp (min)

Hold (min)

Fan

1. Heating 2. Heating 3. Cooling

1400 800 0

2.00 2.00 0.00

10.00 6.00 10.00

1 1 3

Total reaction time = 20 min, P rate: 2 bar/s, IR: 280 °C, P: 80 bar, drive:rot, stirrer: 3. process maxima: pressure: 80 bar; temp. 265 °C.

Fig. 2. Microwave-mediated supercritical ethanol transesterification mechanism for algal biomass. (R0 is a diglyceride group and R1 is a fatty acid chain).

external standard calibration method was implemented for the final product quantification. Fatty acid ethyl ester peaks related to C16:1, C18:1, C18:2 and C20:5 were calibrated using NIST library and mass spectrum (MS) database. The IR spectra were obtained using a Perkin Elmer Spectrum 400 FT-IR/FT-NIR spectrometer. The samples placed on KBr IR cards and scanned at room temperature (25 ± 2 °C) in the mid-infrared region (4000–400 cm1). Five spectra were taken for each sample, at five scans and 4 cm1 spectral resolution. Thermogravimetric analysis (TGA) of wet algal biodiesel (FAEE) was performed using Perkin Elmer Pyris 1 TGA. The samples were heated from 25 to 1000 °C at constant heating rate of 10 °C/min in an atmosphere of nitrogen and oxygen at a constant purge rate of 20 mL/min at the pan. Thin layer chromatography (TLC) was performed on 250-lm silica gel GF plates (scored 10  20 cm, 250 lm). The developing solvent was hexane/diethyl ether/acetic acid (80:20:1, by vol.). Spots were visualized by spraying with 50% aqueous sulfuric acid solution and charring on a hotplate for 10 min at 150 °C in an air circulating to vent hood.

3. Results and discussion 3.1. Influence of process parameters on biodiesel production from wet algal biomass Higher algal biomass to ethanol ratio (wt./vol.) is required to drive the reaction toward completion at a faster rate and shift the equilibrium toward the product side in non-catalytic supercritical alcohol condition (Song et al., 2008). Fig. 3(A) shows the influence of algae to ethanol ratio varies from 1:6 to 1:15 (wt./vol.) on fatty acid ethyl ester yield (FAEE) at controlled power dissipation levels at a fixed reaction time of 25 min. The application of proper power dissipation control has positive influence on the yield of

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reaction product and could result in greater benefits in terms of energy efficiency (Patil et al., 2012). The maximum temperature and pressure examined for the reaction were in the range of 245– 285 °C and 65–80 bar, respectively. It was observed that the yield of algal biodiesel was considerably low initially at 1:6 (wt./vol.) perhaps due to the mass transfer limitation, phase separation barrier and reduced contact area between triglyceride (neutral lipid) and ethanol. The temperature and pressure recorded (65 bar, 245 °C) for the respective algae to ethanol ratio was just above the critical point of ethanol and might not be the sufficient to accelerate the reaction kinetics at faster rate. The reaction yield was then increased to maximum value of 30.9% (based on the dry basis) at 1:9 (wt./vol.) ethanol ratio. In catalyst-free algal reaction system, a large amount of ethanol by volume is required since it acts as both a ‘solvent’ in effective extraction of lipids from algal biomass and ‘reactant and/or acid catalyst’ for transesterification of lipids with enhanced reaction rate to achieve high biodiesel yield (Vieitz et al., 2010). Along with high concentration of ethanol, high pressure and temperature of the reaction system helps water present in the biomass above 250 °C to solubilize the non-polar organic compounds, including hydrocarbons (Kusdiana and Saka, 2004). Furthermore, water present in the biomass at supercritical ethanol condition acts as a tunable co-solvent which not only accelerates the conversion of fats and algal oils to fatty acid ethyl esters (FAEEs), but also reduces the degradation of fatty acids when compared to the supercritical methanol process. This reduction in the degradation of fatty acids is explained by well-known inhibitory effect of water on the oxidation of lipids which attributed to the bonding of hydroperoxides mechanism (Vieitz et al., 2010). These reasons may be attributed to achieve higher yield at higher ethanol concentration. The yield of ethyl ester is found to be gradually declining beyond 1:9 (wt./vol.) algae to ethanol ratio. The yield of wet algal biodiesel at 1:12 and 1:15 (wt./vol.) were calculated as 28.9% and 26.5%, respectively. At higher levels, an excess ethanol started to interfere with the glycerin separation due to increased solubility, which resulted in lower biodiesel yields. Use of excess alcohol makes the separation of final product energy intensive. This negative behavior can also be attributed to a decrease in critical temperature of the reactant and product mixture which leads to the decomposition of the fatty acids in biodiesel to by-products at high temperatures (Tan et al., 2011) resulted due to microwave irradiation effect. A similar trend was observed in direct conversion of wet algal biomass to FAME under supercritical methanol conditions (Patil et al., 2011). In conventional alkali-catalyzed transesterification, FFAs and water has negative effect on the yield of biodiesel. However, in this wet algae microwave-mediated supercritical ethanol process, presence of water and FFA positively affects the formation of ethyl esters and promotes the mechanisms of non-catalytic supercritical reaction (Vieitz et al., 2010). Reaction time plays a crucial role in supercritical ethanol (SCE) biodiesel production as it can influence the productivity and process economics. Fig. 3(B) shows the influence of the reaction time varies from 10 to 30 min with algae to ethanol ratio of 1:9 and controlled power dissipation levels kept fixed. The FAEE yield was observed to be low around 13.5% at reaction time of 10 min. The shorter reaction times do not provide sufficient interaction of the reactant mixture in this twofold effect of extraction and transesterification reaction. The maximum yield of biodiesel was achieved at 25 min reaction time. In catalyst-free algal biodiesel synthesis, it is necessary to run the reaction for longer time as it allows the transesterification reaction to proceed to completion and results in a higher yield of FAEEs from algal biomass. However, slight decline (around 1.3%) in the algal biodiesel was observed at reaction time higher than optimum. The temperature recorded for maximum reaction time (30 min) was above 280 °C. Higher reaction

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P.D. Patil et al. / Bioresource Technology 139 (2013) 308–315 100%

35 30

A

C22:0

80%

25

C20:5 C20:0

70%

20

% FAEE

FAEE Yield (%)

C24:0

90%

15 10

C18:2 C18:1

60%

C18:0

50%

C16:1

40%

C16:0

30%

C12:0

C14:0 C10:0

20%

5

C8:0

10%

0

6

9

12

0%

15

6

B

15

90%

C24:0

80%

C22:0 C20:5

25

70%

% FA EE

FAEE Yield (%)

12

100%

35 30

9

Wet algae to ethanol (wt./vol.)

Wet algae to ethanol (wt./vol.)

20 15

C20:0

60%

C18:2

50%

C18:0

40%

C16:1

C18:1

C16:0

10 5

30%

C14:0

20%

C12:0

10%

C8:0

C10:0

0%

0 10

15

20

25

30

10

15

20

25

30

Reaction Time (min)

Reaction time (min)

Fig. 3. (A) The influence of algae to ethanol ratio on wet algal biodiesel yield (FAEE); (B) the influence of reaction time on wet algal biodiesel yield (FAEE).

time beyond a particular limit in supercritical alcohol process for vegetable oil may lead to greater losses of unsaturated esters due to degradation reactions at higher operational conditions. At higher temperature, FAEE yield can be hampered due to slow degradation of poly-unsaturated fatty acids in algal lipids through isomerization of the double bond functional group from cis-type carbon bonding (C@C) into trans-type carbon bonding (C@C) (Imahara et al., 2008). In addition, autoxidation of the reactive intermediates (organic substrates) and/or unsaturated fatty acids can be possible during algal FAEE process. Based on the experimental analysis for wet algae biodiesel, the optimal conditions for maximum yield of 30.9% are reported as: wet algae/ethanol (wt./vol.) ratio of around 1:9, reaction time of about 25 min, respectively, at controlled power dissipation levels. The temperature noted at these optimum reaction conditions was around 260 °C. FAEE profiles of wet algal biomass (Fig. 3) shows that compositional change within the product mixtures is minor across the parameter space considered. Trends observed are that as the reaction parameters such as algae to ethanol ratio and time increases, the percentage of C16:0 and C18:0, saturated fatty acid ethyl esters increases until the optimum value of either solvent ratio or reaction period is attained, after which yield values are stable. All these fatty acid ethyl esters can remain stable at higher temperature range up to 320 °C and above. The fatty acid ethyl ester percentages of C16:1, C18:1 found to be slightly decreased after the optimal conditions (1:9 wt./vol., 25 min) of the reactions are reached. The percentage of eicosapentaenoic fatty acid ethyl ester (C20:5), the FAEE with the highest degree of unsaturation and greatest polarity, increases initially and then decreases with the increasing in time and ethanol ratio. The reaction pressure and solvent density of the fluid are contributing factors for such behavior of FAEE yield of polyunsaturated fatty acids (Soh and Zimmerman, 2011). The percentage of saturated FAEEs and unsaturated FAEEs were calculated in the range of 46–52% and 50–55% for wet algal biodiesel (Fig. 3). It has been studied that saturated fatty methyl esters

may be decomposed into low molecular fatty acid methyl ester, 1-alkenes, and n-alkanes. These decomposition sequences are similar to generally accepted radical chain scission processes in polymer pyrolysis. For unsaturated fatty acid alkyl esters, cis/trans isomerization and hydrogenation of the C–C double bond are the predominant reactions (Hee-Yong et al., 2011). The percentage of these extracted FAEEs (C8–C24) has significant influence on the physico-chemical, fuel and thermal, oxidative properties of the biodiesel. The saturated fatty acid ethyl esters with no carbon double bonds (C@C) in the compound exhibits higher freezing points while unsaturated (1–3 or 5 double bonds) ethyl esters shows much lower freezing points. In addition, saturated and monounsaturated FAEEs are more stable in supercritical ethanol conditions than poly-unsaturated FAEEs. In this reaction system, lipids were both extracted from algal biomass solids and transesterified to crude biodiesel. The maximum algal biodiesel yield for wet algal biomass obtained at the optimum conditions ranged from 56% to 65% (based on total lipid content in algal biomass). 3.2. Energy consumption The heat energy supplied and thermal energy associated with sample volume required for algal biodiesel extractive-transesterification process (at optimal conditions) is calculated by following equations (Nuchter et al., 2004):

Q mw ¼ P mw t

ð1Þ

Q th ¼ mcp DT ¼ ðma cpa þ me cpe ÞDT

ð2Þ

where ma and me represent the mass of algae sample and ethanol, respectively, used in the reaction system (in grams); cpa and cpe represent the specific heat associated with algae sample and ethanol, respectively (J/(g°K)); DT is the temperature difference observed for the reaction. Eq. (1) represents the heat energy

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supplied by the microwaves which is given in terms of the power dissipation and the time of exposure. Eq. (2) quantifies the thermal effect caused by the microwave radiation in the sample volume. The heat energy supplied and thermal energy associated with sample volume associated with wet algal biodiesel are 1560 and 35.03 kJ, respectively for the test volume, reaction duration and power dissipation used. From case results presented elsewhere (Patil et al., 2012), high microwave energy efficiency (ratio of Q th =Q mw can be observed when high power microwave energy is used for a large amount of sample volume processed in the same batch of a reaction system for a shorter reaction time. Further, regarding the energy consumption, the following analysis can be considered. It has been reported that 1 kg of algal biodiesel production from wet algae requires 42.3 MJ and the same from dry algae requires 107.3 MJ (Lardon et al., 2009). Energy consumptions involved in different steps are algae culture and harvesting, drying, extraction and oil transesterification. Biodiesel production from the wet algae eliminates the need for expensive drying operations which accounts major portion of the total energy consumption (about 84%) in dry algal biodiesel process. Although, the advantages of catalyst free supercritical transesterification reaction for biodiesel production are apparent; there are concerns regarding the huge energy required to conduct supercritical reaction at elevated temperature and pressure exemplified by high algae to alcohol molar ratio at industrial scale production. The process improvements to the microwave-mediated supercritical transesterification can be possible by the addition of co-solvents/catalyst to reduce the severity of the reaction parameters and amount of solvent used in the process (Glišic et al., 2009; Muppaneni et al., 2012). In scale-up point of view, the large scale production of algal biodiesel is feasible if the existing direct transesterification method is converted to continuous flow model coupled with power co-generation unit. The recovery and reuse of unreacted alcohol and value-added utilization of glycerol separated from biodiesel is also essential. 3.3. Determination of nutrients and high value bio-products in algal biomass We have determined the nutrients in both fresh algal biomass and the lipid-extracted algae after the in situ extraction and transesterification process using the established methods for analyzing nutrients in food and other biomass (Osborne and Voogst, 1978). The preliminary report for fresh and LEA is summarized as: crude protein (CP), 36.6% and 30.1%; acid detergent fiber (ADF), 24.5% and 17.1%; neutral detergent fiber (NDF), 39.6% and 29.25%; total digestible nutrients (TDN), 87.4% and 72.05%; fat (EE), 3.92% and 2.04%, respectively. It was observed that the nutrient values of both fresh and the LEA are high enough to be considered as a potential animal feed. More in-depth studies are needed to verify these preliminary results. Some of the high value bio-products that can be extracted and analyzed by GC–MS from N. salina microalgae are phycobiliproteins carotenoids (e.g. indole, oxalic acid, naphthalene); polyunsaturated fatty acids (e.g. EPA, DHA, arachidonic acid); and vitamins (e.g. ascorbic acid, alpha-tocopherol). Other valuable compounds found in algae are vaccenic acid and stigmastan-3,5-diene. Omega-3 fatty acids, eicosapentanoic acid (EPA) and decosahexaenoic acid (DHA) which can be purified to provide a high-value food supplement and can play a vital role in medicine (Patil et al., 2007). 3.4. TEM analysis of algal biomass and lipid extracted algae (LEA) Fig. S1 in the supporting information shows the wet algae sample, before (A) and after (B) microwave extractive-transesterification. In

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(A), virtually all circular profiles of individual cells bound by cell walls contain a large electron-dense, lipid bodies (arrows) that occupy more than 50% of the cytoplasmic areas. Following extractivetransesterification (B), the circular profiles of residual cells are empty and devoid of lipid except for electron density in the circular cell walls. Scale bars measure 1 lm. It was found that the cell walls remain intact with other organelles without the obvious presence of lipid globules, suggesting that the lipid was extracted effectively and converted to FAEE, leaving a residue of cell walls and assorted organelles under the influence of microwave induced supercritical transesterification. Fig. S1(B) also evidenced that no any apparent fragmentation or dissolution of cell walls was resulted due to the high temperature and pressure biomass reactions for extended periods of time.

3.5. Analysis of algal biodiesel The major fatty acid ethyl esters (biodiesel) found in N. salina detected by GC–MS analysis were identified as: C14:0, C16:0, C16:1, C18:1n9c, C18:2n6c, C18:3n3 and C20:5n3 (Fig. 3). Palmitic acid, C16:0 and palmitoleic acid, C16:1 ethyl esters contribute around 70–75% of total fatty acid ethyl esters. This analysis could be helpful for the evaluation of the degradation percentage of the fatty acids as this sample contains major proportion of palmitic acid, C16:0, which is assumed not liable to degradation, considering its high stability (He et al., 2007). It has been observed that under supercritical ethanol (350 °C), a lower percentage of fatty acid decomposition was verified compared to supercritical methanol process (Vieitz et al., 2010). From the GC–MS, it was observed that the algal biodiesel contains olefins, fatty alcohols, aldehydes, sterols and vitamins in minor quantities. The Jet fuel range hydrocarbons such as decane, dodecane, tetradecane and hexadecane were also detected from the analysis. A low percentage of ethyl esters with carbon chain of >18 carbons guarantee a low viscosity for the biodiesel. The percent purity of the FAEE samples using peak areas in GC–MS revealed that this process is comparatively better than conventional and microwave catalytic processes to extract high quality product which in turn indicates the efficient separation and purification of the product. The various absorption peaks of wet algal biodiesel with their group attribution, vibration type and absorption intensity were listed in Table 2. The strong ethyl ester peaks around 1740–1745 cm1 (C@O ester) and 1160– 1170 cm1 (C–O ester) are clearly present in the spectra for wet algal biodiesel (Fig. S2(A)). Outside these two regions, another characteristic peak that indicates the presence of CH3 group in the ethyl ester samples can be observed around 1460 cm1. The band corresponding to the mC(@O)–O vibration shows a peak around 1230–1235 cm1 in algal biodiesel and is one of the confirmations of the conversion of algal lipid to ethyl esters. Fig. S2(C)(a) shows the initial development of the TLC plate for two test results (replicates) of same experimental run of wet algal biodiesel sample where monoglycerides and diglycerides start to appear and Fig. S2(C)(b) presents the results for silica-gel plate showing the position of ethyl esters, triglycerides, fatty acids, tocopherols and sterols found in N. salina algal sp. Two replicates of same experimental run were selected to check the consistency and performance of TLC run as well as to examine the error in process yields for wet algal biodiesel. The low yield samples were selected for TLC analysis for wet algae FAEE for clear visualization of the different products produced during the reaction. The retention factor for ethyl ester and triglycerides was calculated as Rf,EE = 5/ 6.2 = 0.80 and Rf,TG = 4/6.2 = 0.64, respectively. Tocopherols and sterols spots analyzed by the TLC experiment are good natural anti-oxidants.

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Table 2 The various absorption peaks of wet algal biodiesel. Wet algal biodiesel wavenumber (cm1)

Group attribution

Vibration type

Absorption intensity

3354.35 3009.19 2924.76 2853.87 1743.07 1459.30 1165.08 1042.90 723.42

–OH @CH –CH2 –CH2 –C@O –CH3 C–O C–O–C –CH2

Stretching Stretching Asymmetric stretching vibration Symmetric stretching vibration Stretching Shear type vibration Symmetric stretching vibration Anti-symmetric stretching vibration Plane rocking vibration

Weak Weak Strong Strong Strong Middling Strong Weak Weak

3.6. Thermal and oxidative stability of ethyl esters Thermal stability of ethyl esters from algal biomass was determined from onset temperature of thermal degradation under nitrogen atmosphere. The onset temperature for volatilization of wet algal biodiesel was recorded around 125–135 °C. The sample weight loss (thermal degradation) of 10%, 50% and 80% to the initial weight was recorded at the temperature of 179, 290 and 364 °C, respectively. These temperatures can be referred as ‘distillation temperature’ of the ethyl ester samples. TGA curve of ethyl esters of wet algal biodiesel samples represent the three stage decomposition at 120–125, 125–350 and 350–950 °C (Fig. S2(B)). Oxidative stability is the quality indicative parameter for ethyl esters. Fig. S2(B) shows the oxidative degradation TGA profile of their respective ethyl esters performed in oxygen atmosphere under same conditions. The onset temperature of oxidative decomposition for wet FAEE recorded as 115–125 °C. The oxidative degradation temperatures corresponding to the sample weight loss of 10%, 50% and 80% to the initial weight was recorded at 169 °C, 273 °C and 333 °C, respectively. It has been observed that the oxidation temperature of a biodiesel is inversely proportional to the amount of bis-allylic hydrogens. From Fig. S2(B), four steps were observed; being the first one related to oxidation and decomposition reactions and the last steps related to the subsequent polymerization and combustion reactions. It was observed from MSTIC data that wet algal biodiesel retains some natural antioxidants found in algae like sterols, vitamin E includes tocopherols and tocotrienols and oxalic acids in minor quantity which is good indication to ensure better fuel properties. 4. Conclusions An environmentally friendly and energy efficient in situ transesterification process for biodiesel production from wet algae was developed to address many issues in the production of algal biofuels. This microwave-mediated supercritical ethanol transesterification process improves algae extraction efficiency, reduces extractive-transesterification time, and increases biodiesel yield. This non-catalytic transesterification route greatly simplifies the downstream processes, and could potentially address the scaleup limitations of commercial algal biodiesel production if it is developed as a continuous process. However, further investigation on this integrated technology is warranted for scale up of process design, reaction kinetics and thermodynamics, optimization and fuel analysis of biodiesel. Acknowledgements We gratefully acknowledge financial support from US Department of Energy (DE-EE0003046), US Air Force Research Laboratory (FA8650-11-C-2127) and National Science Foundation (EEC-1028968), and NMSU College of Engineering ‘‘Freeport MacMoran Water Lab’’ for the analytical support. The authors are

thankful to Solix Biofuels Inc. for providing the algae samples and Sundar Ponnusamy, Yingqiang Sun, Peter Dailey for their assistances to this project.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.04. 045.

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