Transesterification of Intracellular Lipids Using a Single Step Reactive-Extraction

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1 Transesterification of Intracellular Lipids Using a Single Step Reactive-Extraction Daniel Nelson 1, Ron Sims 1, Sridhar Viamajala 2 1, University of Toledo 2

2 Microorganism derived biodiesel Accumulated as intracellular lipids % of cell lipid content Type of fatty acid (C14,C16, C18) How/what/where to obtain biomass Algae, fungi, etc? Autotrophic, heterotrophic? s/image/algae.jpg Pond, photo-bioreactor?

3 How to get the biodiesel out? Extraction-Transesterification Chemical (Bligh and Dyer) Mechanical Followed by transesterification Other Super critical fluid (SFE), microwave assisted In-situ transesterification img170/5990/palmeb8.jpg Reduces steps/time

4 Objective Derive a model using known reaction mechanisms to describe the in-situ transesterification of TAG s to FAMEs that is scale independent and can be used for large scale production

5 Organism Selection Schizochytrium limacinum SR 21 (ATCC MYA 1381), marine fungus High Lipid Content: 40-50% (dry basis), grow on glycerol Table 1. Fatty Acid Methyl Ester Composition of S. limacinum SR21 Fatty Acid Fraction: (% w/w) of Total Lipid 14:0 15:0 16:0 22:5, 22:6 Reference Myristic Pentadecanoic Palmitic DPA + DHA Yokochi et al., Chi et al., nr This Study 3.8± ± ± ±2.5 DPA = Docosapentaenoic acid, DHA = Docosahexaenoic acid, nr = not reported

6 Concentration of FAME (mg/ml) Concentration of FAME (mg ml -1 ) In-situ reaction In-situ transesterification reaction experiments: Studied significant factors, Acid & Biomass conc mg/ml 125mg/ml 200mg/ml 250mg/ml 20 5% % % % Time (min) Time (min)

7 Model Development Using the fundamental reaction mechanism: Meher 2006 We establish the following identities: S = TAG S = Methanol

8 Model Development We derive the rate expressions: Where: [S] = specific stable TAG concentration at any time during the reaction (mg-tagml -1 Methanol [H + ] = specific stable H + concentration at any time during the reaction (mg- H + ml -1 -Methanol [S ] [SH + ] [SS H + ] = is assumed to be constant throughout the reaction = specific stable TAG-H + complex at any time during the reaction (mg- TAG-H + ml -1 - Methanol = specific stable TAG-H + -MeOH complex at any time during the reaction (mg- TAG-H + -MeOH ml -1 - Methanol

9 Model Development Since the first 2 reactions are assumed to be reversible, the equilibrium constants can be written as: And:

10 Model Development Through substitution of variables, and solving for overall H+ balances, we can reassign the constant terms to single variables V m and k m : Under our conditions, S was much greater than S, and is assumed to be constant. Also, V m and k m can be treated as constant when using a fixed acid conc. In terms of these constants, the rate equation for fatty acid formation can be written as:

11 Data Modeling Fatty acid production measured over time as a function of biomass V m = 1.43 mg ml -1 min -1 k m = mg ml -1

12 Model Verification Since k m is independent of acid concentration, V m was determined at various acid concentrations. From the expression of V m, this parameter should be directly proportional to the initial acid conc.

13 Conclusions An accurate model was developed to describe the in-situ transesterification reaction based on the known reaction mechanism The model thus developed is scale-independent and may be applied to the design of large scale reactors

14 Acknowledgments Sridhar Viamajala, Biological Engineering, University of Toledo Ronald Sims, Biological Engineering, Biological Engineering Program,

15 References Bligh E, Dyer W. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology (1959) 37: No. 8, Carrapiso A., Garcia C. Development in Lipid Analysis: Some New Extraction Techniques and in situ Transesterification. Lipids (2000) Vol. 35, no11, Lewis T, Nichols P, McMeekin T. Evaluation of extraction methods for recovery of fatty acids from lipidproducing microheterotrophs. Journal of Microbiological Methods (2000) 43: Yokochi T, Honda D. Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21. Appl. Microbiol. Biotechnol. (1998) 49: Meher, L.C., Sagar D. Vidya, Naik S.N. Technical aspects of biodiesel production by transesterification- a review. Renewable and Sustainable Energy Reviews. (2006) Vol. 10 Issue 3, Mortia E., Kumon Y. Docosahexaenoic acid production and lipid body formation in Schizochytrium limacinum SR21. Marine Biotechnology (2006) 8; Chi Z, Pyle D, Wen Z, Frear C, Chen S. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochemistry (2007) 42: Pyle D, Garcia R, Wen Z. Producing docosahexaenoic acid (DHA)-rich algae from biodiesel-derived crude glycerol; Effects of impurities on DHA production and algal biomass composition. J. Agric. Food Chem. (2008) 56;

16 Thank You. Questions?

17 GC Chromatograms TAG s extracted from biomass, internal standard FAMEs, converted from TAG s, no TAG remains after in-situ transesterification

18 Model Development

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