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Biodiesel production from heterotrophic microalgal oil
, Qingyu Wu
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China b Department of Biological Sciences, Ningde Teachers College, Fujian, Ningde 352100, PR China Received 6 September 2004; received in revised form 14 February 2005; accepted 6 April 2005 Available online 4 June 2005
Abstract The present study introduced an integrated method for the production of biodiesel from microalgal oil. Heterotrophic growth of Chlorella protothecoides resulted in the accumulation of high lipid content (55%) in cells. Large amount of microalgal oil was eﬃciently extracted from these heterotrophic cells by using n-hexane. Biodiesel comparable to conventional diesel was obtained from heterotrophic microalgal oil by acidic transesteriﬁcation. The best process combination was 100% catalyst quantity (based on oil weight) with 56:1 molar ratio of methanol to oil at temperature of 30 °C, which reduced product speciﬁc gravity from an initial value of 0.912 to a ﬁnal value of 0.8637 in about 4 h of reaction time. The results suggested that the new process, which combined bioengineering and transesteriﬁcation, was a feasible and eﬀective method for the production of high quality biodiesel from microalgal oil. Ó 2005 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel; Transesteriﬁcation; Microalgal oil; Heterotrophic; Chlorella protothecoides; Acid catalyst
1. Introduction Fatty acid methyl esters originating from vegetable oils and animal fats are known as biodiesel. Biodiesel fuel has received considerable attention in recent years, as it is a biodegradable, renewable and non-toxic fuel. It contributes no net carbon dioxide or sulfur to the atmosphere and emits less gaseous pollutants than normal diesel (Lang et al., 2001; Antolin et al., 2002; Vicente et al., 2004). There are four primary ways to make biodiesel, direct use and blending, microemulsions, thermal cracking (pyrolysis) and transesteriﬁcation (Ma and Hanna, 1999). The most common way is transesteriﬁcation as the biodiesel from transesteriﬁcation can be used directly or as blends with diesel fuel in diesel engine (Peterson et al., 1991; Zhang et al., 2003). Biodiesel, primarily rapeseed methyl ester, has been in commercial use as
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an alternative fuel since 1988 in many European countries (Lang et al., 2001). However, in spite of the favourable impact that its commercialization could provide, the economic aspect of biodiesel production prevents its development and large-scale use, mainly due to the high feed cost of vegetable oil (Antolin et al., 2002; Lang et al., 2001). Biodiesel usually costs over US$0.5/l, compared to US$0.35/l for normal diesel (Zhang et al., 2003). Exploring ways to reduce the high cost of biodiesel is of much interest in recent biodiesel research, especially for those methods concentrating on minimizing the raw material cost. Microalgae have been suggested as very good candidates for fuel production because of their advantages of higher photosynthetic eﬃciency, higher biomass production and faster growth compared to other energy crops (Milne et al., 1990; Ginzburg, 1993; Dote et al., 1994; Minowa et al., 1995). Heterotrophic growth of some microalgae has been used for eﬃcient production of biomass and some metabolites such as lipid (Shi et al., 2000, 2002; Wen et al., 2002), which can reduce
0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.04.008
2004). The alga was grown autotrophically and axenically in batch cultures under 26 °C (±1) with continuous illumination at intensities of 40 lmol mÀ2 sÀ1. 70:1 and 84:1) and three diﬀerent temperatures (30. methanol and concentrated sulfuric acid. more the reaction was completed (Al-Widyan and Al-Shyoukh. palm oil and rapeseed oil (Siler-Marinkovic and Tomasevic.1 g/l. Lipid content in heterotrophic cells reached as high as 55. value of microalgal oil (8. cooled. most of the research has concentrated on biodiesel production from vegetable oil such as soybean oil. 1A) but also accumulation of high lipid content in cells.12 g (about 0. 2002. Therefore.1.. protothecoides resulted in not only the disappearance of chlorophyll in cells (Fig. In the present study. There is no information available regarding the production of biodiesel from heterotrophic microalgal oil. The culture medium and method were as described before (Wu et al. Aeration was provided by bubbling air at regular pressure. 2.20% (Table 1). 1992). 50 and 90 °C) were used in the experiments. 2. The molecular weight of the oil was calculated from saponiﬁcation and acid value. Experiments were designed to determine eﬀections of catalyst quantity. 2. Chlorella protothecoides is a microalgae that can be photoautotrophically or heterotrophically grown under diﬀerent culture conditions. For the heterotrophic growth of C. ﬂash point. 2002). six levels of molar ratio of methanol to oil (25:1. Lipid compounds (referred as microalgal oil) was prepared by pulverization of heterotrophic cell powder in a mortar and then extraction with n-hexane. Q.842 X. Texas.01 mol) of microalgal oil was used in all experiments. and left to separated into two layers in a separating funnel. protothecoides was provided by the Culture Collection of Algae at the University of Texas (Austin. USA). 60% and 100% H2SO4 based on oil weight). and washed with petroleum ether and then washed with hot water (50 °C) until the washing were neutral. Lang et al. 2002).3 mg KOH/g) and acid value (8. Heterotrophic growth of C. The characteristics of acidic transesteriﬁcation of microalgal oil and the properties of the biodiesel were investigated. an integrated method for biodiesel production from heterotrophic microalgal oil by acidic transesteriﬁcation was developed.97 mg KOH/g) of the microalgal oil were determined according to the method of Vicente et al. The speciﬁc gravity of the upper layer was precisely measured. 1. which was . In order to investigate the changes of the product speciﬁc gravity with reaction time. Cells were harvested by centrifugation and washed with distilled water and then dried by a freeze dryer.2. Wu / Bioresource Technology 97 (2006) 841–846 the cost of microalgal biomass production and microalgal oil production.1. molar ratio of methanol to oil. The upper oil layer (biodiesel) was separated. heterotrophic growth of C. 30:1. 1998. Reaction conditions Preliminary experiments indicated that the basic catalyst (alkali catalyst) was not suitable for the transesteriﬁcation of microalgal oil probably because of the high acid 3. Acidic transesteriﬁcation of microalgal oil The acid catalyzed transesteriﬁcation were carried out in ﬂasks and heated to the reaction temperature on a shaker or constant temperature bath. protothecoides. The elemental compositions of biodiesel were determined by a CE-440 elemental analyzer. The reaction mixture was heated for the speciﬁed period. Miao. (2004). However. Al-Widyan and Al-Shyoukh. sunﬂower oil. viscosity. The average molecular weight of the oil was 933. 50%.. 2.3. Biodiesel yield (wt. solidifying point and heating value were measured. Results and discussion 3. 1994). Heterotrophic microalgal oil As shown in Fig. A standard reaction mixture consisted of oil. 2001. The details of the culture of heterotrophic cells were reported in our previous research (Wu et al. Lower values of the speciﬁc gravity indicated that more of the heavy glycerine was removed.%) relative to the weight of microalgal oil was estimated. Each 9...4.. protothecoides were measured as described before (Miao et al. Microalgae and sample preparation The strain of C. cold ﬁlter plugging point. 10 g/l glucose was added to the basal medium and the glycine was reduced to 0. protothecoides results in high production of biomass and accumulation of high lipid content in cells. four levels of the catalyst quantity (25%. Methods 2. Antolin et al. 56:1. 45:1. The biodiesel product was obtained by evaporating the ether solution. the acid catalyst (sulfuric acid) was used in the research. aliquots of samples were taken from the reaction mixture at speciﬁed times and centrifuged to obtain the upper layer. The main chemical components of autotrophic and heterotrophic C. The saponiﬁcation (189. time and the temperature in reaction on the yield and properties of biodiesel product. According to the methods of Al-Widyan and Al-Shyoukh (2002) and Siler-Marinkovic and Tomasevic (1998).97 mg KOH/g). Analysis of biodiesel from microalgal oil The properties of biodiesel such as density.
protothecoides cells. 0. . The lipid-soluble compounds from the autotrophic cells appeared in a blackish green with chlorophyll and carotenoid as the major components. whereas the lipid-soluble compounds from the heterotrophic cells appeared in a state of light yellow grease. protothecoides Component (%) Protein Lipid Carbohydrate Ash Moisture Others AC 52. 2.04 1.36 ± 0. Developing high lipid content microalgae or Ôbioengineering microalgaeÕ would be a new and the promising way for biodiesel production in the future. (For interpretation of the references in colour in this ﬁgure legend. protothecoides results in high production of biomass and accumulation of high lipid content in cells. Heterotrophic growth of C. (C) The cells of heterotrophic C. protothecoides under autotrophic (left.88 0. cetane acid by hydrolysis.61 0 20 A 40 60 80 Temperature (oC) 25% ‘Catalyst quantity’ 50% 60% 100% 100 0. 50%.28 15.16 10.64 ± 0.05 5.895 Specific gravity 0.43 ± 0. the high cost of biodiesel due to the high cost of vegetable oil prevents its large-scale use and development.20 ± 0.93 ± 0.885 0. linoleic acid. heterotrophic growth can be performed in conventional microbial bioreactors.65 HC 10. 50 °C and 90 °C. 1992).96 ± 0. C.%) 80 60 40 20 0 Table 1 Contents of the main chemical components of cells of autotrophic (AC) and heterotrophic (HC) C. (B) Almost no lipid vesicles were observed in autotrophic C. green) and heterotrophic (right.57%) (Table 1).39 ± 0. 3. Biodiesel. Q.86 0 20 40 60 80 Temperature (oC) 100 B Fig. The inﬂuence of catalyst quantity and temperature on the yield (A) and the speciﬁc gravity (B) of biodiesel product. yellow) culture conditions.89 0.26 14. The heterotrophic cells were full of lipid vesicles.62 ± 0.87 0.2. At diﬀerent levels of catalyst 25% ‘Catalyst quantity’ 50% 60% 100% 100 Biodiesel yield (wt.. 1.28 ± 0.9 about four times that in autotrophic cells (14. esteriﬁcation and gas chromatographical analysis (Wu et al. which could be easily observed under diﬀerential interference microscopy (Fig. Temperature: 30 °C.57 ± 0. which will improve the yield of biomass and reduce the cost of microalgal biomass production and microalgal oil production. protothecoides under diﬀerential interference microscopy. The fatty acid composition of the oil mainly composed oleic acid. (A) Growth of the cells of C. Miao.X.17 5. 2001). In addition.02 11. It is important as the low cost is desirable for commercial application in biodiesel production.875 0. (B. Large amount of microalgal oil was eﬃciently extracted from these heterotrophic cells by using n-hexane. 160 rpm. 1C). 2. protothecoides is a microalgae that can be photoautotrophically or heterotrophically grown under diﬀerent culture conditions. Eﬀect of temperature and catalyst quantity Fig. Reaction conditions: 30:1 molar ratio of methanol to oil.14 6.42 ± 0. the reader is referred to the web version of this article.. The catalyst quantity based on oil weight: 25%. C) Cells of autotrophic and heterotrophic C.865 0. 5 h of reaction time. primarily made from vegetable oil is being commercialized in many countries (Lang et al. which were mainly lipid compounds (referred as oil). 60% and 100%.) The yield and the speciﬁc gravity of biodiesel product under diﬀerent temperature and catalyst quantity (on oil basis) are shown in Fig. Wu / Bioresource Technology 97 (2006) 841–846 843 However.10 55. protothecoides were full of lipid vesicles.20 ± 0.04 10.
it took about 5–7 h to reach the asymptotic A 0. It may be noticed that at any given level of methanol.878 and 0. The level of 30:1 also gave the low product speciﬁc gravity (0. 160 rpm. 7 h of reaction time. 4.87 0. high catalyst concentrations (2. an excess of methanol in large quanti- . 2B). Wu / Bioresource Technology 97 (2006) 841–846 100 80 60 40 20 0 25:1 30:1 45:1 56:1 70:1 84:1 quantity. However. 3 shows the product yield and speciﬁc gravity under diﬀerent molar ratio of methanol to oil after 7 h of reaction time. Therefore. At the same temperature. Reaction conditions: 30 °C.89 0. 3.91 30:1 ‘Molar ratio’ 45:1 56:1 Specific gravity 0. the process combination of 100% catalyst quantity with temperature of 30 °C would be best. The speciﬁc gravity of the product varied with reaction time.87 0. above conditions were applied to the following experiments. High yields of biodiesel were obtained at molar ratio levels of 45:1 and 56:1 (Fig. whereas the yield was lowest at these levels.92 0.25 M or 100% H2SO4) could produce biodiesel with low speciﬁc gravity in much short time. 3A). The molar ratio of methanol to oil at level of 56:1 resulted in the lowest terminal product speciﬁc gravity and in the shortest reaction time (4 h). Fig. As reported by Al-Widyan and Al-Shyoukh (2002) and Siler-Marinkovic and Tomasevic (1998). Eﬀect of molar ratio of methanol to oil From the results presented in Fig.86 0 1 2 3 4 5 6 7 8 Reaction time (h) Fig. the changes of the product speciﬁc gravity with reaction time were further investigated from these three molar ratio levels as shown in Fig. This is probably because that high temperature and high concentration of H2SO4 could burn some of oil.878). 2A). The yield (A) and the speciﬁc gravity (B) of biodiesel product with diﬀerent molar ratio of methanol to oil. 3. The variation of product speciﬁc gravity with reaction time under diﬀerent molar ratio of methanol to oil. 3B). 0. On economic basis. 2B). 4). low values of speciﬁc gravity were obtained at levels of 100% catalyst quantity (Fig. values of product speciﬁc gravity (Fig. The biodiesel product yields at temperature of 30 °C and 50 °C were 56% and 58% respectively (Fig. Therefore. 2B). 2002). At 45:1 and 30:1 levels.873 and 0. The presence of excess methanol in the transesteriﬁcation process is essential for it is responsible for breaking the glycerine–fatty acid linkages (Al-Widyan and Al-Shyoukh. good quality of biodiesel could be obtained in the presence of 100% acid catalyst at a higher temperature. there were only slight diﬀerence in the yields of biodiesel and their speciﬁc gravity at temperature of 30 °C and 50 °C. 100% catalyst quantity based on oil weight. respectively. whereas low values of speciﬁc gravity were all obtained at 90 °C (Fig. they accounted for 68% and 63%. 2.91 0. 4). The values of the product speciﬁc gravity at these two molar ratio levels were as low as 0.875 respectively (Fig. At level of 100% catalyst quantity. 2A). 100% catalyst quantity and the temperature of 30 °C were used in the experiments of the eﬀects of molar ratio of methanol to oil on the yield and the speciﬁc gravity of biodiesel product.3.88 0.90 0. 100% catalyst quantity based on oil weight. Q. In the present work. 4.86 25:1 30:1 45:1 56:1 70:1 84:1 B Molar ratio Fig.864 respectively (Fig.%) Molar ratio Specific gravity 0. the lowest value of speciﬁc gravity (0.862) was obtained at 90 °C in the presence of 100% catalyst quantity. Miao. Production cost in this case is lower than that when H2SO4 was used at high temperature for a long time. high yields of biodiesel products were all obtained at 50 °C (Fig. 160 rpm.89 0. which cause low yield of biodiesel product.88 0. and the values of product speciﬁc gravity were 0.90 Biodiesel yield (wt. In spite of this. Reaction conditions: 30 °C.844 X. the product speciﬁc gravity decreased with reaction time in a roughly exponential fashion (Fig.
Therefore. The biodiesel from microalgal oil showed much lower cold ﬁlter plugging point of À11 °C in comparison with the diesel fuel (Table 2).. It reduced product speciﬁc gravity from an initial value of 0. Minowa.. protothecoides resulted in the accumulation of a large .9 3.. Sawayama. Conclusions The present study introduced an integrated method for the production of biodiesel from heterotrophic microalgal oil. 1999.5–5. could be a feasible and eﬀective method for the production of high quality biodiesel from heterotrophic microalgal oil.912 to a ﬁnal value of 0. The physical and fuel properties of biodiesel from microalgal oil in general were comparable to those of diesel fuel.. 1994.J... Lang.. Bakhshi.. 1855–1857. 249– 252.838 1. protothecoides is a microalgae that can be photoautotrophically or heterotrophically grown under diﬀerent culture conditions. Heterotrophic growth of C. S. Antolin.1 75 À50 to 10 À3. 53–62. Dalai. A. 2002.Z. Liquid fuel (oil) from halophilic algae: a renewable source of non-polluting energy.81 ASTM biodiesel standard 0. M. Biodiesel production: a review. Vicente et al. Energy 3. Fuel 73. Reaney.864 5.9–4. amount of lipid in cells. F.86–0. Wu..0 (Max À6.X. F. 2004) and biodiesel from microalgal oil and ASTM biodiesel standard is shown in Table 2. M.. B. the best process combination of acidic transesteriﬁcation of microalgal oil was 100% catalyst quantity with 56:1 molar ratio of methanol to oil at temperature of 30 °C. 80..0 Min 100 – Summer max 0. winter max < À15 Max 0.. Bioresour.. 2002. Dote. Recovery of liquid fuel from hydrocarbon-rich microalgae by thermochemical liquefaction. Wu / Bioresource Technology 97 (2006) 841–846 Table 2 Comparison of properties of biodiesel from microalgal oil and diesel fuel and ASTM biodiesel standard Properties Density (kg/l) Viscosity (mm2/s. which combined bioengineering and transesteriﬁcation. M.A.. Large amount of microalgal oil was eﬃciently extracted from these heterotrophic cells by using n-hexane. A comparison of these properties of diesel fuel (Ma and Hanna. Technol. cSt at 40 °C) Flash point (°C) Solidifying point (°C) Cold ﬁlter plugging point (°C) Acid value (mg KOH/g) Heating value (MJ/kg) H/C ratio a 845 Biodiesel from microalgal oil 0.. P. Bioresour. Characteristics of biodiesel from microalgal oil To assess the potential of biodiesel as a substitute of diesel fuel. Lipid content in heterotrophic cells reached as high as 55. 3.374 41 1. 83.5 40–45 1. Y. A. Y. N.K. It was also supported by National Key research plan 2004BA411B05 from Chinese Ministry of Science and Technology. viscosity. cold ﬁlter plugging point.I.. T. ﬂash point. V. 2001. solidifying point and heating value were determined. which reduced product speciﬁc gravity from an initial value of 0.O. Technol. S. 1–15. Hertz. Ginzburg. Most of these parameters comply with the limits established by ASTM related to biodiesel quality (Antolin et al. Good quality of biodiesel could be obtained in the presence of 100% acid catalyst (on oil basis) at high temperature. Technol. Al-Widyan and Al-Shyoukh.7) Max 0. 2002. the properties of biodiesel such as density. Biodiesel comparable to conventional diesel was obtained from heterotrophic microalgal oil by acidic transesteriﬁcation.. Q. Antolin et al.. Preparation and characterization of bio-diesels from various bio-oils.20%. A. Renew. Perez.2 115 À12 À11 0. Experimental evaluation of the transesteriﬁcation of waste palm oil into biodiesel. Miao. 253–256.. References Al-Widyan..I. 2001. Bioresour. Ramirez. 70. 111–114. G. S.912 to a ﬁnal value of 0. Tinaut.8637 in about 4 h of reaction time. Developing high lipid content microalgae or Ôbioengineering microalgaeÕ for biofuels production would be becoming a new and the promising way in biofuels production in the future.. 1993. Hanna. 85. C. 2002).5 – – The data about diesel fuel were taken from published literature as indicated in the text..V. Briceno. Lang et al.8637 in about 4 h of reaction time. Castano. 2002. Bioresour.. Yokoyama.. Al-Shyoukh. C.81 Diesel fuela 0. The best combination of factors was 100% catalyst quantity (based on oil weight) with 56:1 molar ratio of methanol to oil at temperature of 30 °C.4. ties slowed down the separation of the two phases produced as in the case of the levels of 70:1 and 84:1 molar ratio. 4. Inoue. Acknowledgements This research was supported by NSFC project 40272054 and NSFC key project of 40332022 to Q.. Ma. X. 1999. Optimisation of biodiesel production by sunﬂower oil transesteriﬁcation.N. Technol.B. The biodiesel from heterotrophic microalgal oil could be a competitive alternative to conventional diesel fuel. The results suggested that the new process.
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