Advances in direct transesterification of algal oils from wet biomass

Accepted Manuscript Review Advances in direct transesterification of algal oils from wet biomass Ji-Yeon Park, Min S. Park, Young-Chul Lee, Ji-Won Yang PII: DOI: Reference:

S0960-8524(14)01521-1 http://dx.doi.org/10.1016/j.biortech.2014.10.089 BITE 14132

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

31 August 2014 17 October 2014 18 October 2014

Please cite this article as: Park, J-Y., Park, M.S., Lee, Y-C., Yang, J-W., Advances in direct transesterification of algal oils from wet biomass, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.10.089

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Advances in direct transesterification of algal oils from wet biomass

Ji-Yeon Parka,*, Min S. Parkb, Young-Chul Leec, Ji-Won Yangb

a

Biomass and Waste Energy Laboratory, Korea Institute of Energy Research, 152 Gajeong-ro,

Yuseong-gu, Daejeon 305-343, Republic of Korea b

Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of

Korea c

Department of BioNano Technology, Gachon University, 1342 Seongnamdae-ro, Sujeong-gu,

Seongnam-si, Gyeonggi-do 461-701, Republic of Korea

*Corresponding author. Tel.: +82 42 860 3041; fax: +82 42 860 3495. E-mail address: [email protected] (J.-Y. Park)

1

ABSTRACT An interest in biodiesel as an alternative fuel for diesel engines has been increasing because of the issue of petroleum depletion and environmental concerns related to massive carbon dioxide emissions. Researchers are strongly driven to pursue the next generation of vegetable oil-based biodiesel. Oleaginous microalgae are considered to be a promising alternative oil source. To commercialize microalgal biodiesel, cost reductions in oil extraction and downstream biodiesel conversion are stressed. Herein, starting from an investigation of oil extraction from wet microalgae, a review is conducted of transesterification using enzymes, homogeneous and heterogeneous catalysts, and yield enhancement by ultrasound, microwave, and supercritical process. In particular, there is a focus on direct transesterification as a simple and energy efficient process that omits a separate oil extraction step and utilizes wet microalgal biomass; however, it is still necessary to consider issues such as the purification of microalgal oils and upgrading of biodiesel properties.

KEYWORDS Wet microalgae, biodiesel, direct transesterification, wet oil extraction, energy balance

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1. INTRODUCTION Microalgae are photosynthetic microorganisms capable of converting, under light conditions, carbon dioxide and water into macromolecules such as oils, polysaccharides, and proteins (Fu et al., 2010). Some microalgae show high oil productivity compared with plants, and they offer the additional advantage of not competing with food crops and utilizing carbon dioxide and wastewater (Li et al., 2008; Schenk et al., 2008). A great deal of research effort has been devoted to biodiesel production from microalgae as an oil source instead of traditional vegetable oils (Im et al., 2014; Kim et al., 2013; Rawat et al., 2013); however, there are various technical and economic obstacles that have to be overcome before industrial-scale production of microalgal biodiesel can take place (Halim et al., 2012a). As well as upstream challenges related to effective large-scale cultivation, the development of effective and economic oil extraction and biodiesel conversion processes from microalgae is also critical for the successful scale-up of the downstream processes (Halim et al., 2012a). The oil extraction step includes cell disruption by mechanical, chemical, or biological methods and oil collection by solvent. Major bottlenecks of oil extraction are that the extraction of internal oils is energetically demanding because the cell walls of some species of microalgae are strong and thick and that the oil extraction yield is negatively affected in case of a wet biomass (Hidalgo et al., 2013). Advanced processes of wet oil extraction have been suggested to overcome these bottlenecks. Extracted microalgal oils are typically converted to biodiesel by transesterification using alcohols and catalysts. Recently, the combination of oil extraction and biodiesel conversion, called direct (in-situ) transesterification, has been studied (Hidalgo et al., 2013). Direct transesterification refers to the conversion of algal oils present in biomass to biodiesel. Here, direct transesterification includes both the esterification of free fatty acid and the transesterification of triglyceride from microalgae. This process simplifies the production process and improves the biodiesel yield compared with conventional extraction because of the elimination of an oil extraction step that incurs oil loss. The reactions are simple and comprise the addition of alcohols, catalysts, and biomass and sometimes co3

solvents (Baumgartner et al., 2013). This study summarizes recent developments in biodiesel conversion from microalgae both by the two-step conventional process including wet oil extraction and microalgal biodiesel conversion and by direct transesterification; examines wet oil extraction methods using solvent, supercritical fluid, surfactant, ionic liquid, nanoparticle, and hydrothermal process; and discusses briefly points for consideration such as energy balance of wet process, the scale-up of direct transesterification, heterogeneous catalysts, and oil refining.

2. OIL EXTRACTION FROM WET MICROALGAE 2.1 Cell disruption of microalgae It has been frequently reported that the oil extraction yield of dry microalgal cells is relatively higher than that of wet microalgal cells (Guldhe et al., 2014). During the oil extraction process, internal oils are excreted into the outside medium by disruption of thick cell walls and the oils are partitioned into hydrophobic solvents, such as hexane and pentane (Halim et al., 2012b). In addition, the formation of a thin water layer between the cell surface and hydrophobic solvents is known to prevent oil extraction from oleaginous microalgae. Cell disruption methods for the harvested wet microalgae that will prevent the increases of energy and costs due to the additional steps of dewatering and drying of microalgal cells are being sought actively (Halim et al., 2012a). Microalgal cell structure is different from that of cyanobacteria (Drachuk et al., 2013). The difficulty in the cell disruption of oil-rich eukaryotic microalgal cells is associated with the thickness and toughness of their cell walls in comparison with those of prokaryotic cells. For example, the cell wall of Nannochloropsis oculata is usually composed of cellulosic polysaccharides with algaenans while that of Chlorella sp. is composed of microfibrils of glucosamine polymers producing a chitinous cell wall and only partially of algaenans, and this results in cell walls of different sizes and morphologies in various microalgae. Consequently, several mechanical and chemical or combinational cell disruption methods have been suggested to facilitate post-oil extraction by solvents from the interior compartments of algal cells, for example, the comparative application of ultrasound, high-pressure homogenization, bead-beating, hydrolysis of polysaccharides by sulfuric acid treatment, 4

and osmotic shocks (Halim et al., 2012b). The cell disruption methods are classified based on the manner in which they achieve microalgal cellular disintegration (Fig. 1). Table 1 summarizes recent developments in oil extraction from various microalgae. The cell disruption method with high oil extraction yield should be accompanied by the selection of microalgae with high oil content. In the next step, these techniques are coupled with transesterification for biodiesel production to enhance the biodiesel conversion yield.

2.2 Organic solvent extraction Many efforts to extract oil effectively had been investigated since the introduction of the Folch method using chloroform-methanol (2:1, v/v) and its modifications. Halim et al. (2012a) compared organic solvent extraction for various solvents. From the relationship between the Hansen dispersion parameter and solubility of triacetin in the solvent, the oil extraction trends of chloroform, methanol, water, chloroform/methanol (1:2, v/v), chloroform/methanol/water (1:2:0.8, v/v/v), hexane, isopropanol, hexane/isopropanol (3:2, v/v), and ethanol were tested. Although chloroform has the ability to dissolve cells for the easy extraction of neutral lipids (acylglycerols and free fatty acids) as well as polar lipids (phospholipids and glycolipids), there is a reluctance to use it for oil extraction because of the toxicity of chlorinated solvent. As an alternative, hydrophobic hexane is frequently suggested owing to its selective extraction in neutral lipids and less toxicity. Its mixture with isopropanol (hexane/isopropanol, 3:2, v/v) exhibited good results for oil extraction, comparable to that of the Folch method using chloroform-methanol for concentrate or disrupted concentrate of microalgae (Halim et al., 2011). This indicates that polar alcohol enhances the extraction of polar lipids in membrane based lipid-protein associations. Soxhlet extraction using hexane could supply fresh organic solvents continuously to microalgal cells, leading to minimal solvent consumption; however heating for reflux requires high energy consumption and suffers from challenges in the scaleup process (Halim et al., 2012a). Halim et al. (2014) reported the sustainability of solvent extraction to extract oil from wet microalga (Tetraselmis suecica). When moisture content was less than 20%, the increase in the 5

moisture content did not significantly reduce the oil extraction yield as long as the mixture remained monophasic (methanol or hexane/isopropanol). Recently, an interesting approach for wet algal samples or cultures has been done on switching solvents with switchable-polarity or -hydrophilicity that recover microalgal oil from the extracting solvent simply by the addition of carbon dioxide (Boyd et al., 2012; Du et al., 2013; Samori et al., 2010, 2013). This approach shows that oil extraction by switchable solvents does not require drying of biomass or distillation of solvents. Until now, this system has been applied for oil extraction from a few species such as Botryococcus braunii, Nannochloropsis gaditana, Tetraselmis suecica, and Desmosesmus communis.

2.3 Application of ultrasound, microwave, and supercritical process As mentioned above regarding cell disruption, mechanical force such as ultrasound, high-pressure homogenization (HPH), pulsed electrical fields (PEF), and microwave offer synergistic and enhanced oil extraction yields (Natarajan et al., 2014). Gerde et al. (2012) monitored the intracellular material release of Schiazochytrium limacinum and Chlamydomonas reinhardtii from disruption by free radicals formed by ultrasound treatment with energy input of 800 J/10 ml. To prevent free radicalinduced degradation of oil, careful optimization of the sonication conditions is required. In comparison with ultrasound extraction (18.9% lipid/g DCW), a higher oil extraction yield (28.3% lipid/g DCW) has been shown from microwave oil extraction of Scenedesmus sp. with 91.8% biodiesel yield, indicating that microwave cell disruption is efficient (Guldhe et al., 2014; Koberg, et al., 2011). Grimi et al. (2014) compared different cell disruption methods for Nannochloropsis sp., including PEF, high voltage electric discharge, ultrasound, and HPH, indicating that HPH disruption was the most effective. PEF treatment increased an oil extraction yield of Scenedesmus sp. 3.1-fold (30.6/33.7 kWh/m3) (Lai et al., 2014). Callejon et al. (2014) examined the cell disruption effects of Nannochloropsis gaditana on HPH. An oil extraction yield was 10.9% lipid/g DCW at 9 L/h and 1700 bar. Souza Silva et al., (2014) tested four different pretreatment methods for cell disruption: ultrasound, microwave, autoclave, and electroflotation by alternating current (EFAC). EFAC promotes the formation of oxidant species (O3, H2O2, and OH radical) which may effectively act on cell 6

disruption. The oil extraction yields were 33.7% (microwave), 24.8% (EFAC), 15.4% (autoclave), and 13.3% (ultrasound), respectively. Meanwhile, osmotic shock with Chlamydomonas reinhardtti led to efficient cell disruption followed by increased oil recovery, but a high salt concentration requires posttreatment (Yoo et al., 2012). Supercritical fluid extraction does not use solvents, but supercritical conditions are necessary (Taher et al., 2014). Supercritical carbon dioxide (SCCO2) extraction is widely employed at the supercritical region above its critical point (Pc = 72.9 atm and Tc = 31.1˚C) (Halim et al., 2011). In particular, the SCCO2 process has selectivity for the specific extraction of acylglycerols with minor co-extraction of polar lipids and free fatty acids as well as a very short oil extraction time because of the rapid penetration of SCCO2 through the cellular matrices. This process shows non-reactivity with oil and there is no need for solvent removal and post-distillation. SCCO2 as a green solvent showed improved oil extraction efficiency and decreased pigment, nitrogen, and phospholipid in the produced biodiesel (Soh and Zimmerman, 2011). The feasibility of scale-up for biodiesel production from Chlorella protothecoides using supercritical fluid extraction was examined for future potential (Tabernero et al., 2012). Oil extracted by subcritical ethanol extraction from wet paste Tribonema minus was converted catalytically to biodiesel through acid-alkaline transesterification (Wang et al., 2013).

2.4 Use of surfactants, ionic liquids, nanoparticles for cell destabilization Cell destabilizers have been used to facilitate oil extraction from wet microaglae. Ions, biomolecules, and nanoparticles are known to affect the weakening of cell walls. Huang and Kim (2013) reported a cationic surfactant-based harvesting and cell disruption method where cetyltrimethylammonium bromide (CTAB) induced 100% oil extraction in wet microalgae of 80% moisture content, achieving an energy-intensive method in the downstream process of oil production. Yoo et al. (2014) examined cell disruption using tertiary-amine cations deposited on polydimethylaminomethylstyrene (pDMAMS) film by initiated chemical vapor deposition (iCVD) and obtained a cell disruption yield of 25.6%. This process also has the potential to remove chlorophyll. 7

Alternatively, ionic liquid (1-ethyl-3-methylimidazolium methylphosphate, [Emim][MeO(H)PO2]) dissolves wet and saliferous microalgae at room temperature without heating. After water addition, the microalgae are precipitated and [Emim][MeO(H)PO2] is recovered for reuse, while oil is easily extracted from the precipitated microalgae (Fujita et al., 2013). Lee et al. (2013a) studied cationic aminoclays for destabilization of Chlorella sp. KR-1 to enhance the oil extraction yield. Additionally, the introduction of hydrogen peroxide (H2O2) with attachment of the aminoclays onto the wet cells generated powerful oxidants, free OH radicals, and these disrupted Chlorella sp. KR-1 resulting in an oil extraction efficiency of 26.7% lipid/g DCW. Oil droplets were observed with 100% biodiesel conversion (Lee et al., 2013b). Moreover, aminoclay conjugated TiO2 was applied for harvesting and simultaneous cell disruption of Chlorella sp. KR-1 (Lee et al., 2014). Although the cells were partially disintegrated, there was no increase in oil extraction efficiency by hexane after aminoclay conjugated TiO2 treatment because of the weak damage to the cell walls. In case alcohols are added as a co-solvent, the oil extraction efficiency can increase because polar alcohol is able to reduce the interfacial energy between the thin water layer of the algal cells and the solvent media. The Fenton’s reactant (an aqueous solution of H2O2 and FeSO4 ) was used to disrupt cells. The reaction between H2 O2 and Fe2+ produces OH radicals which in turn may attack and degrade the cell walls. The oil extraction yield of 17.3% lipid/g DCW was obtained from Chlorella vulgaris (90% moisture content) (Steriti et al., 2014). Huang et al. (2014) examined the performance of pressure-assisted ozonation (PAO) to disrupt cells of Chlorella vulgaris. PAO causes both chemical and mechanical damage to cells resulting in an oil extraction yield of 27%. In short, the ionic properties of additives used during oil extraction from wet microalgal cells are effective for increasing the efficiency of oil extraction; however, the cost of the additives, their recyclability, and toxicity for humans are crucial factors for industrial biorefinery.

2.5 Hydrothermal treatment With a heating process, the oil extraction yield from a wet microalga, Chlorella vulgaris, increased to 337.4 mg/g cells using an acid catalyzed hot-water treatment with a 1% H2SO4 concentration 8

heated at 120˚C for 60 min (Park et al., 2014a). Meanwhile, an oil extraction yield of 472.4 mg/g cell was obtained from Aurantiochytrium sp. and 141.7 mg/g of docosahexaenoic acid (DHA) was extracted under the optimal condition of 1.0% H2SO4 concentration, 100˚C, and 30 min (Choi et al., 2014). They also investigated the effect of an anionic surfactant, sodium dodecyl benzene sulfonate (SDBS). A combination of SDBS with H2SO4 showed a high yield of extracted oils from wet Chlorella vulgaris in the hot-water process, and at 2.0% H2SO4 and 0.2% SDBS the content of free fatty acids in the oil was 96.1% and the oil extraction yield was 266.0 mg/g cell (Park et al., 2014b). The dosage of acid catalyst for esterification of the free fatty acids was reduced significantly in the free fatty acids-rich microalgal oil. In addition, Im et al. (2014) achieved a biodiesel conversion yield of 91% of from wet Nannochloropsis oceanica using chloroform, methanol, and H2SO4 in a single pot process, thereby improving the oil conversion yield and eliminating the drying cost. Also, a 79% extraction yield of transesterifiable oils was obtained from a mixture of Chlorella and Scenedesmus sp. with 84% moisture content through wet lipid extraction by acid and alkaline hydrolysis at 90˚C and ambient pressure (Sathish and Sims, 2012). By adding 0.5 M sulfuric acid solution, chlorophyll was removed effectively through precipitation. Cell disruption was performed by using steam explosion at 120˚C and 180˚C for 5 min. The explosion caused by a sudden release of pressure disrupts the cellular structure of microalgae, making oil more accessible. The oil extraction yields of 21.4% at 120˚C and 22.0% at 180˚C were obtained from Nannochloropsis gaditana (83% moisture content), respectively (Nurra et al., 2014). Lee et al. (2014) applied hydrothermal nitric acid treatment to extract oil effectively from wet Nannochloropsis salina. The maximum oil yield of 24.4% was obtained using 0.57% nitric acid at 120˚C for 30 min. Hydrothermal liquefaction includes a heating step by hot and compressed water or supercritical water under high pressure (e.g., 300˚C and 8.6 MPa) and produces biocrude oils from wet algal biomass (Yeh et al., 2013; Savage, 2012). Also it may be used with a variety of heterogeneous catalysts such as Pd/C, Pt/C, Ru/C, Ni/SiO2-Al2 O3, CoMo/γ-Al2O3, and zeolites; the use of nonprecious metal catalysts may advance the feasibility of the process (Duan and Savage, 2011). At temperatures of 300-350˚C, hydrothermal liquefaction is suitable for the generation of viscous 9

biocrude oil with heating values of 60-80% and rich in heteroatoms, i.e., 3-5% of N and 8-10% of O. Thermal and catalytic processes should be followed in order to remove the heteroatoms and upgrade the biocrude oil so that it is free-flowing at room temperature. At higher temperature (400-600˚C) and higher pressure (25 MPa), wet microalgal biomass is converted to usable gaseous products. Recently, microwave assisted hydrothermal pyrolysis at 180-210˚C and below 300 psi has been studied as an efficient oil extraction method from wet microalgal biomass without hydrolysis of triglycerides (Budarin et al., 2012). In this process, the addition of salt increased the hydrolysis of carbohydrates and protein leading to fractionation to the water phase. A pilot plant test has been carried out to produce biocrude oil by continuous hydrothermal processing of Chlorella and Spirulina strains at various temperatures (250-350˚C), residence times (3-5 min), and pressures (150-200 bar) (Jazrawi et al., 2013).

3. DIRECT TRANSESTERIFICATION 3.1 Conventional transesterification process Transesterification of microalgal oils for biodiesel production has been carried out by both homogeneous and heterogeneous catalysis. Homogeneous alkaline catalysis has been the most used route for biodiesel production because it catalyzes the reaction at low temperature and atmospheric pressure and also a high conversion yield can be achieved in a short time. Sodium hydroxide (NaOH) and potassium hydroxide (KOH) are widely used as alkaline catalysts; however, alkaline catalysts cause the free fatty acids in oils to produce soap and are not suitable for microalgal biodiesel production because of the high free fatty acid content in microalgal oils. Acid catalysts overcome the limitation of high free fatty acid content and are typically used when the content of free fatty acids is higher than 1%. The most used acid catalysts are sulfuric acid (H2SO4) and hydrochloric acid (HCl). They require larger response times and higher temperature than alkaline catalysts (Hidalgo et al., 2013; Vonortas and Papayannakos, 2014). In some studies, initially, an acid catalyst is used to convert free fatty acid into esters through esterification. After the free fatty acid content in the oils is reduced to less than 1%, a second transesterification step for the oils is performed by using an alkaline catalyst. 10

Despite the high conversion yields reached by homogeneous catalysts, there is always catalyst loss after the reaction. In this sense, the use of heterogeneous catalysts is expected to play a relevant role in future because of their advantages of recovery and reuse. Umdu et al. (2009) reported the transesterification of oils from Nannochloropsis oculata by using CaO and MgO supported on alumina with the highest yield (97.5%) obtained by loading CaO on Al2O3. Microalgal oils extracted from Dunaliella tertiolecta and Nannochloropsis oculata were converted to biodiesel by metallic oxides composed of ZrO, TiO, and Al2O3 that simultaneously esterified and transesterified free fatty acids and triglycerides under supercritical conditions, and the final conversion yield reached 85% (Krohn et al., 2011). Enzymes can be used to transform microalgal oils to biodiesel. Li et al. (2007) reported a 98% biodiesel conversion yield from oils extracted from Chlorella protothecoides using immobilized lipase from Candidiasis sp. Lai et al. (2012) used an ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate, [Bmim][PF6]) and an organic solvent (tert-butanol) as a medium for enzymatic transesterification of oils from Chlorella pyrenoidosa. Two immobilized lipases, Penicillium expansum lipase (PEL) and Candida antarctica lipase B (Novozym 435), produced higher yields of 90.7% and 86.2% respectively in [Bmim][PF6] compared with those of 48.6% and 44.4% respectively in tert-butanol. Tran et al. (2012) reported immobilized enzymatic transesterification of disrupted microalgal biomass for Chlorella vulgaris ESP-31 with 97.3% biodiesel conversion yield. Some technologies applied for oil extraction such as ultrasound, microwave, and supercritical process can be applied again in the transesterification step to enhance the conversion yield of biodiesel (Hidalgo et al., 2013; Patil et al., 2011). A comparison of an alternative direct transesterification and the conventional process is shown in Fig. 2. In direct transesterification, microalgal biomass is directly transesterified using alcohol and catalyst in one reactor. The oil extraction and transesterification are carried out in one step with a direct conversion of oil-bearing biomass to biodiesel, thereby avoiding the steps of cell disruption and oil extraction from biomass. Some pretreatments of microalgae, except for conventional oil extraction using solvents, have been performed before direct transesterification to enhance the reaction yield. 11

The direct transesterification methods are classified based on the manner in which they achieve effective transesterification (Fig. 3). Table 2 summarizes recent developments in direct transesterification from various microalgae. Wet microalgal biomass was effectively converted to microalgal biodiesel through direct transesterification.

3.2 Direct transesterification process Several studies have been conducted using dry microalgal biomass to investigate direct transesterification processes. Dry biomass reacts with sulfuric acid and methanol. Methanol acts both as an extraction solvent and an esterification reagent. The use of an additional solvent such as hexane or chloroform helps the easy extraction of oils within microalgal cells and enhances the contact of microalgal oils with the esterification reagent (Cao et al., 2013). Sathish et al. (2014) discussed the effects of water inhibition on direct transesterification and concluded that: (1) the formation of fatty acid methyl ester (FAME) is a reversible reaction and water can hydrolyze biodiesel back to methanol and free fatty acids, (2) water contained within the biomass can shield oils from the extracted solvent and prevent oils from being brought into the reaction, and (3) the acid catalyst can be deactivated owing to water competing for available protons in the reaction. Although water inhibits the transesterification of wet microalgal biomass, an economic process using wet biomass should be developed. Several wet oil extraction technologies have been developed recently and these methods can be combined with a direct process to overcome water inhibition. Transesterification of Chlorella sp. and Nannochloropsis oculata was performed at moisture contents of 0%, 1.5%, and 10% using acid and alkaline catalysts (sulfuric acid, sodium hydroxide, and sodium methoxide). Sulfuric acid as a catalyst showed a higher FAME yield of 73% for Nannochloropsis oculata and 92% for Chlorella sp., and the yield was not dependent on the salinity of the biomass and decreased with the increase of moisture (Velasquez-Orta et al., 2013). Direct transesterification of mixed microalgal biomass for biodiesel production was reported with an increased biodiesel yield obtained because of esterification of fatty acids from membrane phospholipids as well as transesterification of triglycerides (Wahlen et al., 2011). Biodiesel conversion 12

yield was proportional to methanol loading and inversely proportional to water content. Im et al. (2014) reported direct transesterification of wet microalgae, Nannochloropsis oceanica, with a 65% moisture content and obtained a high conversion yield of 91.1% under 0.2 g cell, 0.3 g H2SO4, 2 ml chloroform, and 1ml methanol at 95˚C for 90 min. To enhance the yield, a chloroform-methanol mixture (2:1, v/v) was applied as a solvent and reaction reagent. Cao et al. (2013) carried out direct biodiesel production from Chlorella pyrenoidosa (90% moisture content). Hexane was used as an additional solvent and the maximum biodiesel yield was 92.5% under 0.1 g cell, 0.5M H2SO4, 8 ml hexane, and 4 ml methanol at 120˚C for 180 min. For direct transesterification, a high level of methanol and sulfuric acid is required compared with a commercial biodiesel process. The amount of methanol and sulfuric acid should be reduced to avoid the need for a large reactor and reactor corrosion by sulfuric acid. Solvents such as pentane and diethyl ether have been used to reduce the volume of methanol by enhancing the reaction yield. These solvents assist in the extraction of microalgal oils in conjugation with methanol by improving the diffusion of the microalgal oils across the cell walls. This is facilitated by increasing the selectivity and solubility of the extraction media, thereby providing greater availability of the oils for the transesterification process (Ehimen et al., 2012). In a one-step reaction using Schizochytrium limacinum (80% moisture content), chloroform (4 ml)-methanol (3.4 ml) showed a higher biodiesel yield compared with methanol (7.4 ml) only (Johnson and Wen, 2009). Although this indicates the possibility of reducing the amount of methanol, further development is necessary to reduce the amount of solvent. Through a combination of sonication and co-solvent using Chlorella sp., the molar ratio of oil to methanol decreased markedly. After 2 h ultrasound agitation, the FAME conversion yield was the same at a molar ratio of 1:105 as at 1:210 and 1:315. After 8 h mechanical stirring with diethyl ether, there was a good conversion yield at a molar ratio of 1:79. After 2 h with a combination of the above two methods, there was a conversion yield of 99.9% at a molar ratio of 1:52 (Ehimen et al., 2012).

3.3 Advanced approaches of direct transesterification process 13

To enhance the conversion yield of direct transesterification, the application of microwave or ultrasound that can enhance the mass transfer rate between immiscible phases, simultaneously diminishing the reaction time, was suggested (Hidalgo et al., 2013). In microwave-assisted transesterification, methanol absorbs microwave radiation, quickly redirecting its dipole. This rearrangement allows the destruction of the methanol-oils interface. The microwaves transfer energy in an electromagnetic form, and the oscillating microwave field tends to move continuously to the polar ends of molecules or ions. Consequently collisions and friction between the moving molecules generate heat. Heat is transferred directly into the reaction media with a rapid temperature increase throughout the sample (Hidalgo et al., 2013). Rapid heating leads to localized high temperature and pressure gradients, which assist in cellular wall degradation and enhance mass transfer rates (Patil et al., 2011). Microwave with 800W power was applied to Nannochloropsis sp. and a FAME conversion yield of 80.1% was obtained (Patil et al., 2011a). Chlorella pyrenoidosa (moisture content 80%) was mixed with methanol, chloroform, and sulfuric acid; and the biodiesel production yield through a onestep process using microwave were 6-fold and 1.3-fold higher respectively than with a two-step process using conventional heating (Cheng et al., 2013). Ultrasound is an effective method to enhance the mass transfer rate between immiscible phases (Hidalgo et al., 2013); therefore, it improves transesterification yields and reduces reaction times. The oil-alcohol phase boundary is disrupted because of the collapse of ultrasonically induced cavitation bubbles (Ehimen et al., 2012). The cavitation bubbles produced by ultrasound also attack the microalgal cell walls enhancing oil extraction from cells. Through a combination of sonication and co-solvent using Chlorella sp., the FAME conversion yield reached 99.9% (Ehimen et al., 2012). Although these processes enhance the FAME yield or reaction rate, it is still necessary to decrease the costs of these technologies including equipment installation. To enhance the transesterification yield, the application of supercritical conditions was suggested (Patil et al., 2011b). Supercritical methanol without catalyst and with additional solvent produced FAME from Nannochloropsis sp. (moisture content 90%) with the conversion yield increased to 85.8% and polar phospholipids were converted to FAME as well as free fatty acid and triglyceride (Patil et 14

al., 2011b). In another approach, it was shown that when triglycerides were hydrolyzed to free fatty acids by an anionic surfactant, SDBS, at low pH during the microalgal oil extraction process, the amount of sulfuric acid used as an esterification catalyst could be reduced markedly (Park et al. (2014b). For direct transesterification, the application of a surfactant as an additive can reduce the dosage of sulfuric acid. Some pretreatments, except for conventional oil extraction using solvents, were performed before direct transesterification to enhance the conversion yield. After cell walls of Chlorella vulgaris ESP31 (moisture content 86-91%) were disrupted by sonication, direct enzymatic transesterification was performed with methanol, hexane, and immobilized lipase and the yield of FAME increased to 95.7% (Tran et al., 2013). Levine et al. (2010) applied a supercritical method to the system: first, Chlorella vulgaris (80% moisture content) was hydrolyzed by supercritical water at 250˚C; second, supercritical direct transesterification was performed using ethanol without catalyst; and, at 325˚C, the biodiesel yield increased to 100%. In addition, wet microalgal biomass was pretreated with a short chain alcohol in order to remove excess water, which inhibited the transesterification. During the second step, an alcohol and a catalyst were added to the pretreated biomass for esterification at mild conditions less than 120˚C. Water was removed from the alcohols used in both steps, after which both the solvent and catalyst could be reused (Yoo et al., 2012, 2014). In most studies, the transesterification catalyst was sulfuric acid because microalgal oils contain high free fatty acid content. Few researchers used alkaline catalysts for direct transesterification. Koberg et al. (2011) used SrO as a solid alkaline catalyst with Nannochloropsis for biodiesel production and showed a high biodiesel conversion yield. Patil et al. (2011a) also used an alkaline catalyst, potassium hydroxide (KOH), for direct transesterification of Nannochloropsis sp., and the FAME conversion yield was 80.1% with 2% catalyst. Free fatty acids form soap with alkaline catalysts, and this makes difficult the separation of biodiesel and alcohol layers. Consequently, acid catalysts are typically used for microalgal oil that contains at least 1% free fatty acid (Huerga et al., 2014); however, alkaline catalysts may be suitable for the direct transesterification of microalgae because oils structured in microalgae are mostly triglyceride form. 15

4. ENERGY BALANCE OF WET AND DRY PROCESSES Since the majority of the research results are derived from small laboratory scale experiments and from batch mode reactions rather than continuous process, it is rather difficult to calculate and compare the economics of various procedures. However, it is clear that the energy requirement is still substantial for the majority of the processes that employ dry biomass. Lardon et al. (2009) reported an analysis of the potential impact of biodiesel production from microalgae. They compare nominal fertilizing or nitrogen starvation and dry or wet extraction. For Chlorella vulgaris, by nitrogen starvation, oil content increased to 38.5% from 17.5% although the growth rate decreased. Wet oil extraction significantly reduces heat requirements, but the lower extraction yields erode slightly the benefit of this technique. The total energy consumptions for 1kg biodiesel production were 106.4, 41.4, 48.9, and 19.9 MJ for normal-dry, normal-wet, low N-dry, and low N-wet respectively. Net energy balances were -2.6, 105, 12, and 66 MJ for normal-dry, normalwet, low N-dry, and low N-wet respectively; therefore, wet extraction is the desirable direction for commercialization. If the advanced wet oil extraction methods mentioned above are used in the analysis, there will be a remarkable increase in net energy due to the increased oil extraction yield from wet biomass. Xu et al. (2011) compared the energy balance of the dry and wet routes for biofuel production from microalgae. The drying process in the dry route and the oil extraction process in the wet route consume a significant amount of energy. The analytical results indicate that the wet route has more potential for producing biofuel. Sills et al. (2013) also agreed with the need for wet oil extraction. For economic feasibility, they suggested the recovery of nutrients from waste streams and the production of high energy co-products such as methane derived by anaerobic digestion. For the wet biomass process, energy analyses of one-step and two-step microalgal biodiesel production will be developed soon. Analyses on direct transesterification combined with advanced wet oil extraction technologies will provide valuable information for evaluating the process feasibility of microalgal biorefinery. 16

5. FUTURE PROSPECTS Biodiesel production from microalgae demands high energy consumption during the cell disruption, oil extraction, and biodiesel conversion processes. The application of direct transesterification could be an alternative that reduces the critical steps to produce biodiesel, eliminates the oil extraction process, and simultaneously reduces the amount of necessary equipment. The most important issue in microalgal biodiesel production is the utilization of wet biomass. Because dewatering and drying of microalgae needs excess energy consumption, advanced oil extraction methods showing high yield under wet conditions have to be developed. The oil extraction methods mentioned above should be combined with a direct transesterification process to enhance the reaction yield and to reduce the amount of solvent and sulfuric acid. After processes to lower the solvent dosage and reduce the reactor volume have been determined, the scale-up issue can be discussed. While wet extraction process eliminates the amount of energy and costs required for drying of the microalgal biomass, this process still require a large amount of energy that is associated with thermal input for heating and cooling of the reactor. Therefore, the extraction or direct conversion process needs to be developed for their operational efficiency at lower temperatures and pressure. Any procedure that can be performed below 100˚C will contribute positively to the costs associated with reactor design, energy requirement, operational and maintenance costs of reactor. There is a need for homogeneous catalysts to be replaced by heterogeneous catalysts to enable reuse and for the design of a continuous process using heterogeneous catalysts. A purification process for microalgal oils is another challenging step that incurs further additional costs compared with easily purified plant oils. Activated carbon or activated clay cause high oil loss from microalgal oils. There is a need to develop an efficient method for purification because the extracted oils contain large quantity of pigments, which makes conversion to biodiesel difficult. Typically, plant or microalgae based biodiesel has poor oxidation stability and poor cold temperature properties, and its properties should be upgraded to the level of those of petroleum diesel in consideration of its ultimate end use. To upgrade biodiesel properties, triglycerides and free fatty acids of microalgal oils are converted to 17

long-chain normal paraffins by the catalytic hydrotreatment. These paraffins have a number of carbon atoms close to the corresponding range of diesel fuel. Meanwhile, considering the soft nature of microalgae compared to lignocellulosic biomass, it may be feasible to adopt a thermochemical process for the production of microalgal biocrude oils instead of selective extraction or conversion of lipid portion to biodiesel (Barreiro et al., 2013; Frank et al., 2013). Thermochemical process can be a convenient and flexible platform for biofuels production because of no need of solvent and sulfuric acid. If that is the case, the overall costs of fuel produced may be either comparable to or lower than those of direct transesterification process. For example, hydrothermal liquefaction at subcritical temperature can produce biocrude oils from microalgal biomass. The biocrude oils undergo hydrocracking and are further refined within the traditional petroleum refinery infrastructures to produce green diesel, gasoline and aviation fuels, and other chemical feedstock for producing plastics and other renewable chemical products. The various technical breakthroughs are still necessary for catalytic upgrading and further purification of various chemical feedstock. These include the development of cheap or recyclable catalysts or a continuous process for thermochemical conversion, and analytical capability to characterize biocrude asphaltene that has not been well studied so far. These advancements will eventually make the microalgal refinery a reality. The wet extraction or direct transesterification for the production of microalgal biodiesel will continue to evolve to achieve better energy balance, sustainability, capital cost and operating cost. The evolution of these processes will not only affect the costs associated with the downstream oil production, but also positively influence other upstream processes such as microalgal cultivation and harvest strategy. Once all of these positive gains from both upstream and downstream processes are combined, microalgal biofuels will be one step closer to achieving price parity with the petroleum derived transportation fuels.

6. CONCLUSIONS Recent studies on direct transesterification for microalgal biodiesel production from wet biomass 18

were reviewed. The crucial parameters for direct transesterification are the moisture content of the biomass, the amount of solvent and sulfuric acid, and the final yield of FAME. The results of recent development in microalgal oil extraction show a high oil extraction yield despite the use of wet biomass. If the recently developed methods that include effective additives or hydrothermal treatment are combined with direct transesterification, microalgal biodiesel will be closer to a commercial application. Other considerations in biodiesel production from microalgae such as the oil purification to remove pigments, the use of heterogeneous catalysts, and upgrading of biodiesel properties require further study.

ACKNOWLEDGEMENTS This work was supported by the New & Renewable Energy Technology Development Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Knowledge Economy (No. 20123010090010) and by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2012M3A6A2053880).

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26

Table 1 Methods and results summary of recent studies investigating microalgal oil extraction Cell disruption method

Microalgal species

State of microagal biomass

Conditions

Oil extraction yield (%)a

Reference

Organic solvent

Tetraselmis suecica

20% moisture content

Methanol Hexane/isopropanol (3:5, v/v)

16.1 12.4

Halim et al., 2014

100˚C for 10 min at 1000W

18.9

Scenedesmus sp.

5% cells in chloroform/ethanol (1:1, v/v) 10% cells in chloroform/ethanol (1:1, v/v)

Ultrasound

Microwave Pulsed electrical fields High-pressure homogenization Electroflotation by alternative current Autoclave Osmotic shock

Guldhe et al., 2014 15 kHz for 2 min at 100W

28.3

Scenedesmus sp.

Harvested cells

30.6 kWh/m3 (24˚C to 54˚C)

33.0

Lai et al., 2014

Nannochloropsis gaditana

86% moisture content

9 L/h and 1700 bar

10.9

Callejon et al., 2014

Mixed culture

>99% moisture content

12V and 5A (~1.5 kHz)

33.7

100˚C for 10 min

15.4

Souza Silva et al., 2014

>99% moisture content

NaCl (60 g/L)

34.5

Yoo et al., 2014

400 mL/min CO2 flow rate 4.9-14.1 min residence time 60˚C and 30 MPa

7.1

Halim et al., 2011

100 rpm for 18 h

25.6

Yoo et al., 2014

1% H2O2 at 300 rpm for 3 h

26.7

Lee et al., 2013b

Chlamydomonas reinhardtii

Supercritical carbon dioxide

Chlorococcum sp.

~80% moisture content

Functional polymeric membrane Aminoclay-based

Aurantiochytrium sp. KRS101 Chlorella sp. KR-1

14.6 g/L cell concentration 20 g/L cell concentration 27

H2O2 Fenton’s reactant

90% moisture content

0.5M H2 O2 and 0.024M FeSO4 for 3 min

17.3

Steriti et al., 2014

Pressure-assisted ozonation Hot water Hot water

Chlorella vulgaris

Microalgae suspension

Ozone (~1.5% by volume)

27.0

Huang et al., 2014

Chlorella vulgaris Aurantiochytrium sp.

20 g/L cell concentration 50 g/L cell concentration

33.7 47.2

Hot water

Mixed culture

84% moisture content

1% H2SO4 at 120˚C for 60 min 1% H2SO4 at 100˚C for 30 min 1M H2SO4 and then 5M NaOH (90˚C for 30 min)

Park et al., 2014a Choi et al., 2014 Sathish and Sims, 2012

Steam explosion Hot water a

Chlorella vulgaris

Nannochloropsis gaditana Nannochloropsis salina

10.9

83% moisture content

180˚C and 10 bar for 5 min

22.0

Nurra et al., 2014

20 g/L cell concentration

0.57% HNO3 at 120˚C for 30 min

24.4

Lee et al., 2014

Oil extraction yield (%) = weight of extracted oil (g) / dry cell weight (g) * 100

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Table 2 Methods and results summary of recent studies investigating direct transesterification Transesterification method

Microalgal species

State of microagal biomass

Conditions

Biodiesel conversion yield (%)

Reference

Acid catalyst

Chlorella sp.

0% moisture content 1.5% moisture content 10% moisture content

60˚C for 19 h Oil:H2SO4:methanol= 1:0.35:600 (m/m/m)

92.0 80.0 61.0

Velasquez-Orta et al., 2013

Acid catalyst and cosolvent

Nannochloropsis oceanica

65% moisture content

91.1

Im et al., 2014

Acid catalyst and cosolvent

Chlorella pyrenoidosa

90% moisture content

92.5

CaO et al., 2013

Microwave

Chlorella pyrenoidosa

80% moisture content

10.5a

Cheng et al., 2013

Supercritical methanol

Nannochloropsis sp.

90% moisture content

85.8

Patil et al., 2011b

Enzyme after sonication

Chlorella vulgaris ESP-31

86-91% moisture content

95.7

Tran et al., 2013

Supercritical method

Chlorella vulgaris

80% moisture content

100 (58.7 FAEE)

Levine et al., 2010

a

0.2g cell, 0.3g H2SO4, 2mL chloroform, and 1mL methanol (95˚C for 90 min) 0.1g cell, 0.5M H2SO4, 8mL hexane, and 4mL methanol (120˚C for 180 min) 1g cell, 0.2g H2SO4, 4mL chloroform, and 4mL methanol (500W for 40s) Cell:methanol=1:9 (w/v) 255˚C for 25 min 10 min sonication Oil:methanol=1:94.9 (m/m) 45˚C for 48 h 250˚C for 45 min (water) and 325˚C for 120 min (ethano1)

Biodiesel yield (% of dry biomass)

29

Fig. 1. Classification of cell disruption methods.

30

(a)

(b)

Fig. 2. Conventional process (a) and direct transesterification process (b) for microalgal biodiesel production.

31

Fig. 3. Classification of direct transesterification methods.

32

Highlights ● Recent developments in wet oil extraction and biodiesel conversion from microalgae ● Direct transesterification omitting a separate oil extraction step for wet microalgae ● Needs for purification of microalgal oils and upgrading of biodiesel properties

33