(334b) Viable and Non Viable Microalgae Fractionation Employing Insulator-Based Dielectrophoresis

Dielectrophoresis (DEP) is a transport mechanism driven by polarization effects on particles in the presence of non uniform electrical fields. DEP can be employed effectively to concentrate and fractionate microorganisms non-destructively.¹ To produce the non uniform fields to perform DEP, the most common approach is to apply AC electric fields employing arrays of microelectrodes.² However, microelectrode array- based DEP systems face some issues, such as degradation of microlectrodes and high production costs.¹ An alternative to electrode-based DEP is the technique called insulator-based DEP (iDEP), where non-uniform electric fields are produced employing arrays of insulating structures.

It has been demonstrated that differences in cell's membrane state and conductivity has effect on the dielectrophoretic response of cells¹  and that DEP can be used to manipulate live and heat-treated cells of Listeria,³ to separate viable and non viable Yeast cells⁴ and separate live and dead bacteria.¹

Several research groups had proved that the viability of microalgae is a critical factor for the absorption of elements like copper⁵ or tributyltin (TBT)⁶. However, there is not a method to efficiently differentiate live from dead microalgae. The present work shows the capability of iDEP to fractionate and concentrate live and dead microalgae.

Green algae (Ankiristodesmus sp., UTEX 98) has been successfully concentrated and fractionated using an array of cylindrical insulating posts to induce dielectrophoresis under several applied voltages.  

A microchannel made from glass was employed, the channel  was 10.12-mm-long, 1-mm-wide, 10-μm deep, and had an array of 32 cylindrical posts distributed in eight columns of four posts each, posts had a diameter of  200-μm and were arranged in a square array  250-μm center-to-center. The cylindrical posts transverse the entire height of the microchannel (Figure 1).

A sample of 50 ml mid-logarithmic phase (OD₆₇₅ = 1.5) culture of microalgae was divided into two equal parts, which were centrifuged at 3000 rpm for 3 minutes. Both pellets were resuspended in buffer solution (pH 9 σm=100 μS/cm). Dead microalgae cells were prepared by heating an aliquot oc cells 100 ºC for 1 hour, and the reduced viability of this sample was corroborated with epifluorescence microscopy. Fluorescence of SYTOX Green (S7020, Invitrogen, Carlsbad CA, USA) a dye that only penetrates damaged cell membranes, and autofluorescence were observed simultaneously, allowing the discrimination of viable and non viable cells.

A sample of mixed live and heat-treated was introduced at the inlet reservoir of the microchannel. Care was taken to eliminate pressure-driven flow, and then electrodes were placed at the inlet and outlet reservoirs in order to apply a DC electric field.

Several voltages ranging from 100 to 750 V/cm were applied to the sample and differential trapping of live and dead cells was shown by two separate bands of different color. Viable cells (red) are trapped at the wider regions between the circular posts (negative DEP), and non viable cells (green) exhibit less negative DEP, since they are trapped at the narrower regions between the circular posts.

Figure 1. Schematic representation of the microchannel employed with iDEP experimentation.

References

1.       Lapizco-Encinas, B.H., Simmons, B.A., Cummings, E.B., Fintschenko, Y., 2004 a. Dielectrophoretic Concentration and Separation of Live and Dead Bacteria in an Array of Insulators. Analytical Chemistry.  76, 1571-1579.

2.       Washizu, M., Kurosawa, O., 1990.  Electrostatic manipulation of DNA in microfabricated structures. IEEE Transactions on Industry applications Appl. 26, 1165-1172.

3.       Li, H., Bashir, R., 2002. Dielectrophoretic separation and manipulation of live and heat-treated cells of Listeria on microfabricated devices with interdigitated electrodes. Sensors and Actuator A.  86, 215-221.

4.       Markx, G.H., Talary, M.S., Pethig, R., 1994. Separation of viable and nonviable yeast using dielectrophoresis. Journal of Biotechnolgy. 32, 29-37.

5.    Al-fawwaz, A. T. and W. O. Wan-Maznah.  Biosorption of Copper Using Live and Dead Green Microalgae Isolated from Penang Rivers, Malasya. International Conference on Enviormental Research and Technology, Penang, Malasya, Proc. 940-943. 

6.       Tam, N.F.Y., Chong, M.Y., Wong, Y.S.  Removal of tributyltin (TBT) by live and dead microalgal cells. Marine Pollution Bulletin. 45, 362-371.