Abstract
Accurate determination of fatty acid contents is routinely required in microalgal and yeast biofuel studies. A method of rapid in situ fatty acid methyl ester (FAME) derivatization directly from wet fresh microalgal and yeast biomass was developed in this study. This method does not require prior solvent extraction or dehydration. FAMEs were prepared with a sequential alkaline hydrolysis (15 min at 85 °C) and acidic esterification (15 min at 85 °C) process. The resulting FAMEs were extracted into n-hexane and analyzed using gas chromatography. The effects of each processing parameter (temperature, reaction time, and water content) upon the lipids quantification in the alkaline hydrolysis step were evaluated with a full factorial design. This method could tolerate water content up to 20 % (v/v) in total reaction volume, which equaled up to 1.2 mL of water in biomass slurry (with 0.05–25 mg of fatty acid). There were no significant differences in FAME quantification (p > 0.05) between the standard AOAC 991.39 method and the proposed wet in situ FAME preparation method. This fatty acid quantification method is applicable to fresh wet biomass of a wide range of microalgae and yeast species.
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References
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917
Carrapiso AI, Garcia C (2000) Development in lipid analysis: some new extraction techniques and in situ transesterification. Lipids 35(11):1167–1177
Chakraborty M, Miao C, McDonald A, Chen SL (2012) Concomitant extraction of bio-oil and value added polysaccharides from Chlorella sorokiniana using a unique sequential hydrothermal extraction technology. Fuel 95(1):63–70. doi:10.1016/j.fuel.2011.10.055
Chisti Y (2008) Biodiesel from microalgae beats bioethanol. Trends Biotechnol 26(3):126–131. doi:10.1016/j.tibtech.2007.12.002
Christie WW (1993) Advances in Lipid Methodology: v. 2. Oily Press, Dundee:69–111
Dong T, Wang J, Miao C, Zheng Y, Chen S (2013) Two-step in situ biodiesel production from microalgae with high free fatty acid content. Bioresource Technol 136(0):8–15. doi: 10.1016/j.biortech.2013.02.105
Ehimen EA, Sun ZF, Carrington CG (2010) Variables affecting the in situ transesterification of microalgae lipids. Fuel 89(3):677–684. doi:10.1016/j.fuel.2009.10.011
Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226(1):497–509
Griffiths MJ, van Hille RP, Harrison STL (2010) Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids 45(11):1053–1060. doi:10.1007/s11745-010-3468-2
Iverson JL, Sheppard AJ (1975) Programmed temperature gas chromatographic analysis of esters of fatty acids. J Chromatogr Sci 13(10):505–508
Iverson SJ, Lang SLC, Cooper MH (2001) Comparison of the bligh and dyer and folch methods for total lipid determination in a broad range of marine tissue. Lipids 36(11):1283–1287. doi:10.1007/s11745-001-0843-0
Johnson MB, Wen ZY (2009) Production of biodiesel fuel from the microalga schizochytrium limacinum by direct transesterification of algal biomass. Energ Fuel 23:5179–5183. doi:10.1021/Ef900704h
Laurens LML, Quinn M, Van Wychen S, Templeton DW, Wolfrum EJ (2012) Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification. Anal Bioanal Chem 403(1):167–178. doi:10.1007/s00216-012-5814-0
Lee JY, Yoo C, Jun SY, Ahn CY, Oh HM (2010) Comparison of several methods for effective lipid extraction from microalgae. Bioresource Technol 101:S75–S77. doi:10.1016/j.biortech.2009.03.058
Lepage G, Roy CC (1986) Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27(1):114–120
Lewis T, Nichols PD, McMeekin TA (2000) Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs. J Microbiol Meth 43(2):107–116
Li Q, Du W, Liu DH (2008) Perspectives of microbial oils for biodiesel production. Appl Microbiol Biot 80(5):749–756. doi:10.1007/s00253-008-1625-9
Lipke PN, Ovalle R (1998) Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180(15):3735–3740
Liu KS (1994) Preparation of fatty-acid methyl esters for gas-chromatographic analysis of lipids in biological-materials. J Am Oil Chem Soc 71(11):1179–1187. doi:10.1007/Bf02540534
McNichol J, MacDougall KM, Melanson JE, McGinn PJ (2012) Suitability of soxhlet extraction to quantify microalgal fatty acids as determined by comparison with in situ transesterification. Lipids 47(2):195–207. doi:10.1007/s11745-011-3624-3
O’Fallon JV, Busboom JR, Nelson ML, Gaskins CT (2007) A direct method for fatty acid methyl ester synthesis: application to wet meat tissues, oils, and feedstuffs. J Anim Sci 85(6):1511–1521. doi:10.2527/Jas.2006-491
Suter B, Grob K, Pacciarelli B (1997) Determination of fat content and fatty acid composition through 1-min transesterification in the food sample principles. Z Lebensm Unters F A 204(4):252–258. doi:10.1007/s002170050073
Wang G, Wang T (2012) Characterization of lipid components in two microalgae for biofuel application. J Am Oil Chem Soc 89(1):135–143. doi:10.1007/s11746-011-1879-8
Wang RX, Fu Y, Lai LH (1997) A new atom-additive method for calculating partition coefficients. J Chem Inf Comp Sci 37(3):615–621. doi:10.1021/Ci960169p
Yu X, Dong T, Zheng Y, Miao C, Chen S (2014) Investigations on cell disruption of oleaginous microorganisms: hydrochloric acid digestion is an effective method for lipid extraction. European Journal of Lipid Science and Technology:n/a-n/a. doi:10.1002/ejlt.201400195
Zheng Y, Li T, Yu X, Bates PD, Dong T, Chen S (2013) High-density fed-batch culture of a thermotolerant microalga Chlorella sorokiniana for biofuel production. Applied Energy 108(0):281–287 doi: 10.1016/j.apenergy.2013.02.059
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Dong, T., Yu, L., Gao, D. et al. Direct quantification of fatty acids in wet microalgal and yeast biomass via a rapid in situ fatty acid methyl ester derivatization approach. Appl Microbiol Biotechnol 99, 10237–10247 (2015). https://doi.org/10.1007/s00253-015-6909-2
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DOI: https://doi.org/10.1007/s00253-015-6909-2