title: Current lipid extraction methods are significantly enhanced adding a water treatment step in Chlorella protothecoides.
abstract: BACKGROUND
  Microalgae have the potential to rapidly accumulate lipids of high interest for the food, cosmetics, pharmaceutical and energy (e.g. biodiesel) industries. However, current lipid extraction methods show efficiency limitation and until now, extraction protocols have not been fully optimized for specific lipid compounds. The present study thus presents a novel lipid extraction method, consisting in the addition of a water treatment of biomass between the two-stage solvent extraction steps of current extraction methods. The resulting modified method not only enhances lipid extraction efficiency, but also yields a higher triacylglycerols (TAG) ratio, which is highly desirable for biodiesel production.
  
  
  RESULTS
  Modification of four existing methods using acetone, chloroform/methanol (Chl/Met), chloroform/methanol/H2O (Chl/Met/H2O) and dichloromethane/methanol (Dic/Met) showed respective lipid extraction yield enhancement of 72.3, 35.8, 60.3 and 60.9%. The modified acetone method resulted in the highest extraction yield, with 68.9 ± 0.2% DW total lipids. Extraction of TAG was particularly improved with the water treatment, especially for the Chl/Met/H2O and Dic/Met methods. The acetone method with the water treatment led to the highest extraction level of TAG with 73.7 ± 7.3 µg/mg DW, which is 130.8 ± 10.6% higher than the maximum value obtained for the four classical methods (31.9 ± 4.6 µg/mg DW). Interestingly, the water treatment preferentially improved the extraction of intracellular fractions, i.e. TAG, sterols, and free fatty acids, compared to the lipid fractions of the cell membranes, which are constituted of phospholipids (PL), acetone mobile polar lipids and hydrocarbons. Finally, from the 32 fatty acids analyzed for both neutral lipids (NL) and polar lipids (PL) fractions, it is clear that the water treatment greatly improves NL-to-PL ratio for the four standard methods assessed.
  
  
  CONCLUSIONS
  Water treatment of biomass after the first solvent extraction step helps the subsequent release of intracellular lipids in the second extraction step, thus improving the global lipids extraction yield. In addition, the water treatment positively modifies the intracellular lipid class ratios of the final extract, in which TAG ratio is significantly increased without changes in the fatty acids composition. The novel method thus provides an efficient way to improve lipid extraction yield of existing methods, as well as selectively favoring TAG, a lipid of the upmost interest for biodiesel production.
authors.affiliation: Research Laboratory in Applied Metabolic Engineering, Department of Chemical Engineering, École Polytechnique de Montreal, P.O. Box 6079, Centre-ville Station, Montreal, QC, H3C 3A7, Canada.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Ren
authors.affiliation: Research Laboratory in Applied Metabolic Engineering, Department of Chemical Engineering, École Polytechnique de Montreal, P.O. Box 6079, Centre-ville Station, Montreal, QC, H3C 3A7, Canada.
authors.forename: Xinhe
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Université du Québec à Rimouski, 310 allée des Ursulines, Rimouski, QC, G5L 3A1, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Turcotte
authors.affiliation: Université du Québec à Rimouski, 310 allée des Ursulines, Rimouski, QC, G5L 3A1, Canada.
authors.forename: Jean-Sébastien
authors.initials: JS
authors.lastname: Deschênes
authors.affiliation: Université du Québec à Rimouski, 310 allée des Ursulines, Rimouski, QC, G5L 3A1, Canada.
authors.forename: Réjean
authors.initials: R
authors.lastname: Tremblay
authors.affiliation: Research Laboratory in Applied Metabolic Engineering, Department of Chemical Engineering, École Polytechnique de Montreal, P.O. Box 6079, Centre-ville Station, Montreal, QC, H3C 3A7, Canada. mario.jolicoeur@polymtl.ca.
authors.forename: Mario
authors.initials: M
authors.lastname: Jolicoeur
mesh_terms: D056804:Biofuels; D018533:Biomass; D002458:Cell Fractionation / Q000379:methods; D002708:Chlorella / Q000737:chemistry* / Q000166:cytology; D005227:Fatty Acids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D008055:Lipids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D059625:Liquid-Liquid Extraction / Q000379:methods*; D000432:Methanol; D012997:Solvents; D014280:Triglycerides / Q000032:analysis* / Q000302:isolation & purification; D014867:Water
chemical_list: D056804:Biofuels; D005227:Fatty Acids; D008055:Lipids; D012997:Solvents; D014280:Triglycerides; D014867:Water; D000432:Methanol
keywords: Chlorella protothecoides; High TAG ratio; High extraction yield; Lipid extraction; Two-stage solvent extractions; Water treatment