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Page 1: MICROALGAL CULTURE, LIPID PRODUCTION AND EXTRACTION … · MICROALGAL CULTURE, LIPID PRODUCTION AND EXTRACTION USING AN INTEGRATED MICROFLUIDIC ... output reservoir was analyzed by

MICROALGAL CULTURE, LIPID PRODUCTION AND EXTRACTION

USING AN INTEGRATED MICROFLUIDIC SYSTEM H.S. Lim, J.Y.H Kim, H.S Kwak and S.J. Sim

*

Korea University, Korea

ABSTRACT

We present a novel method for simple and efficient lipid analysis by integrating multi-process including microalgal

culture, lipid production and lipid extraction on a single microfluidic device. We devised a double-layered PDMS device,

composed of cell culture chamber and connecting channel filled with micropillar array between culture chamber and

output reservoir. Lipid accumulation was induced by supply of nitrogen deficient media into microchannel and lipid

extraction was performed by alcohol solvents such as 70% ethanol, isopropanol. Cell debris was removed by micropillar

filter and lipid extract from output reservoir was analyzed by thin layer chromatography and gas chromatography.

KEYWORDS: Microfluidics, Microalgae, Culture, Lipid production, Lipid extraction, Micropillar filter

INTRODUCTION

Microalgal lipid has gained increasing interest as a promising alternative to fossil fuels due to its sustainability.

Simple and efficient lipid analysis is required for selection of desirable strains for biofuel production. However,

conventional method for lipid accumulation and extraction requires multiple centrifugation steps and long processing time

for cell harvesting, media exchange, lipid extraction using toxic organic solvents. Microfluidic approaches enable

multiple processes in a single integrated device. Although there have been numerous reports on extraction and detection

of biological molecules such as DNA [1], protein [2], RNA [3] in microfluidic systems, few reports on lipid extraction in

microfluidics [4]. This might be caused by toxic organic solvent used for lipid extraction, which require solvent-resistant

material such as glass [4]. In the present work, we also demonstrated PDMS-based microfluidic device can be applied to

lipid extraction by using alcohol solvents such as ethanol and isopropanol.

EXPERIMENTAL

A double-layered PDMS device was fabricated by soft lithography. The lower layer containing a cell culture chamber

with a diameter of 8 mm and output reservoir was bonded to glass slide after oxygen plasma treatment. The upper layer

was designed to have connecting microchannel filled with micropillar array with 2 µm gaps to filter microalgal cell and

bonded to lower layer after plasma treatment (Fig. 1).

Figure 1: Schematic diagram of integrated microfluidic device

for microalgal culture and lipid extraction

Chlamydomonas reinhardtii (CC-503) was grown in microfluidic culture chamber including TAP (Tris-Acetate-

Phosphate) medium under continuous light at 50 µmol photon/m2/s, 23°C. Cell growth was monitored by measuring

optical density (at 800 nm) using plate reader (Tecan). After 4 days, lipid accumulation was induced by supply of

nitrogen deficient TAP medium into culture chamber. Lipid accumulation was monitored by measuring fluorescent

intensity at the excitation wavelength of 530 nm and emission wavelength of 580 nm after staining cells using a

fluorescent dye, Nile red.

After lipid accumulation, cell lysis and lipid extraction were performed by injecting various alcohol solvents such as

70% ethanol, isopropanol and methanol into microchannel at 60°C using PHD syringe pump (Harvard Apparatus).

Neutral lipid content and fatty acid content in lipid extract obtained from output reservoir was analyzed by thin layer

chromatography (TLC) and gas chromatography.

978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1920 17th International Conference on MiniaturizedSystems for Chemistry and Life Sciences27-31 October 2013, Freiburg, Germany

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RESULTS AND DISCUSSION

As shown in Figure 2, we fabricated double-layered PDMS device composed of cell culture chamber and connecting

channel filled with micropillar array between culture chamber and output reservoir. We evaluated the performance of this

integrated microfluidic system. For the growth of microalgal cells, seed culture was injected into culture chamber

through the inlet. We observed culture chamber is filled with TAP medium including microalgal cells (Fig. 3A) and

micropillar array filter completely block the movement of cells (Fig. 3B).

Figure 2: Integrated microfluidic device fabricated as double-layered PDMS

Figure 3: Image of (A) microalgal cells in culture chamber and

(B) connected microchannel with micropillar filter

We monitored cell growth in culture chamber by measuring optical density using microplate reader and optical

density (A800) increased from 0.2 to 1 for 4 days. After growth for 4 days, culture medium was exchanged with nitrogen

deficient TAP medium to induce lipid accumulation by supply nitrogen deficient medium into culture chamber through the

microchannel. Lipid content was monitored by measuring fluorescence intensity of microalgal cells stained with Nile red

using microplate reader.

When lipid accumulation reached maximum value, alcohol solvents such as 70% ethanol, isopropanol were injected into

culture chamber through microchannel for cell lysis and lipid extraction. In this study, we utilized alcohol solvents to

minimize swelling effect on PDMS. Before extraction in microfluidic device, we analyzed lipid extraction ability of each

alcohol solvent compared to conventional solvent such as chloroform/methanol (2:1, v/v). Although overall fatty acid

content in lipid extract by alcohol solvent was reduced compared to that extracted by chloroform/methanol, the difference

was not significant (Fig. 4A). We found that double-layered PDMS device maintains its structure during extraction process

using alcohol solvent such as 70% ethanol and isopropanol. In addition, cell debris occurred by cell lysis was successfully

removed by micropillar array filter and we obtained transparent lipid extract from integrated device using alcohol solvents

(Fig. 4B). When we analyzed the lipid extract from integrated device by TLC, we observed neutral lipid (TAG) was obtained

using alcohol solvent such as 70% ethanol in PDMS-based microfluidic device (Fig. 4C). Despite simple process, neutral

lipid (TAG) or total fatty acid content obtained from integrated microfluidic system was comparable to that obtained from

conventional Bligh-Dyer method.

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Figure 4: (A) Analysis of fatty acid content in extracted lipid using various solvents, (B) lipid extract from

integrated device using 70% Ethanol and (C) TLC analysis of extracted lipid from (B)

CONCLUSION

Microalgal lipid analysis requires multi-step processes for cell culture, lipid production, and lipid extraction including

multiple centrifugation steps, long processing time and usage of organic solvents. Microfluidic approaches allow per-

forming multiple processes in a single integrated device by simply changing flow of medium and solvents. In conclusion,

we report that integrated microfluidic device developed in this study can provide effective means to develop simple and

efficient process required for lipid analysis.

ACKNOWLEDGEMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea govern-

ment (MEST) (grant no. R0A-2008-000-20078-0/2010-0027955), and the grant from Korea CCS R&D Center (grant no.

2011-0031997) funded by the Ministry of Science and Technology of the Korean government of the Republic of Korea.

REFERENCES

[1] E. A. Oblath, W. H. Henley, J. P. Alarie and J. M. Ramsey, A microfluidic chip integrating DNA extraction and re-

al-time PCR for the detection of bacteria in saliva, Lap on a Chip, 13, pp. 1325-1332, (2013).

[2] A. J. Hughes and A. E. Herr, Microfluidic Western blotting, Proceedings of the National Academy of Sciences USA,

109, pp. 21450-21455, (2012).

[3] M. Tsaloglou, F. Laouenan, C. Loukas, L. G. Monsalve, C. Thanner, H. Morgan, J. M. Ruano-Lópezb and M. C.

Mowlema, Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an inte-

grated microfluidic platform, Analyst, 138, pp. 593-602, (2013).

[4] T. Sun, S. Pawlowski and M. E. Johnson, Highly efficient microscale purification of glycerophospholipids by micro-

fluidic cell lysis and lipid extraction for lipidomics profiling, Analytical Chemistry, 83, pp. 6628-6634, (2011).

CONTACT

*S.J. Sim, tel: +82-2-32904853; [email protected]

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