Transesterification of

Intracellular Lipids Using a

Single Step Reactive-Extraction

Daniel Nelson1, Ron Sims1, Sridhar Viamajala2

Utah State University1, University of Toledo2
 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? http://apexlyo.com/attachment
s/Image/algae.jpg

− Pond, photo-bioreactor?

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 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
http://img170.imageshack.us/
img170/5990/palmeb8.jpg

− Reduces steps/time
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 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

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 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., 1998 2.7 7.6 34.2 51.7
Chi et al., 2007 4.0 nr 52.0 42.0
This Study 3.8±0.11 2.5±0.08 53.9±1.3 40.1±2.5
DPA = Docosapentaenoic acid, DHA = Docosahexaenoic acid, nr = not reported

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 In-situ reaction

 In-situ transesterification reaction experiments:

− Studied significant factors, Acid & Biomass conc.
140
35

120 66mg/ml
30
125mg/ml

Concentration of FAME (mg mL -1 )

Concentration of FAME (mg/mL)

200mg/ml
25 100
250mg/ml

20 80
5%

15 2% 60

1.5% 40
10

1%
5 20

0 0
0 10 20 30 40 50 60 70 80 90 0 20 40 60 80 100 120 140 160 180
Time (min) Time (min)

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 Model Development

 Using the fundamental

reaction mechanism:

Meher 2006

 We establish the
following identities:
– S = TAG
– S’ = Methanol
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 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’] = is assumed to be constant throughout the reaction
[SH+] = specific stable TAG-H+ complex at any time during the reaction (mg- TAG-H+ ml-1- Methanol
[SS’H+] = specific stable TAG-H+-MeOH complex at any time during the reaction (mg- TAG-H+-MeOH
ml-1 - Methanol

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 Model Development

Since the first 2 reactions are

assumed to be reversible, the
equilibrium constants can be
written as:

And:

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 Model Development
Through substitution of variables, and solving for overall H+ balances,
we can reassign the constant terms to single variables Vm and km:

Under our conditions, S’ was much greater than S, and is assumed to

be constant. Also, Vm and km 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:

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 Data Modeling
Fatty acid production measured over time as a function of biomass

Vm = 1.43 mg ml-1 min-1 km = 23.15 mg ml-1

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

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 Acknowledgments

 Sridhar Viamajala, Biological Engineering, University of Toledo

 Ronald Sims, Biological Engineering, Utah State University

 Biological Engineering Program, Utah State University

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 References
Bligh E, Dyer W. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry
and Physiology (1959) 37: No. 8, 911-917

Carrapiso A., Garcia C. Development in Lipid Analysis: Some New Extraction Techniques and in situ
Transesterification. Lipids (2000) Vol. 35, no11, 1167-1177

Lewis T, Nichols P, McMeekin T. Evaluation of extraction methods for recovery of fatty acids from lipid-
producing microheterotrophs. Journal of Microbiological Methods (2000) 43: 107-116

Yokochi T, Honda D. Optimization of docosahexaenoic acid production by Schizochytrium limacinum

SR21. Appl. Microbiol. Biotechnol. (1998) 49: 72-76

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, 248-268

Mortia E., Kumon Y. Docosahexaenoic acid production and lipid body formation in Schizochytrium
limacinum SR21. Marine Biotechnology (2006) 8; 319-327

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: 1537-1545

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; 3933-3939

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Thank You.
Questions?
 GC Chromatograms

 TAG’s extracted
from biomass,
internal standard

 FAMEs, converted
from TAG’s, no
TAG remains after
in-situ
transesterification

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 Model Development

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