Microfluidic Platform for Quantitative Characterization of Biodiesel Formation in Microalgae

Abstract

Microfluidics always has been appealed to biologist because of the capability to control the cellular microenvironment in both a spatial and temporal manner. These system can generate a biological relevant stimulus including concentration gradient, nutrient conditions and physical and chemical stresses by taking advantage of basic characteristics of laminar flow and diffusion. Moreover, microfluidic techniques have been presented a novel paradigm for screening system with their small volume fractions and high-throughputs. Unfortunately, there are several considerable things for applying microalgae to microfluidic system. This research presents microalgae research into microfluidic platform by offering each solutions at every limiting steps. First, quantification of lipid droplet (LD) which is one of the major product of carbon conversion in microalgae is required for developing and optimizing microalgal bioprocess engineering. This report describes new fluorescence probes for LDs staining

Seoul-Fluor (SF) and JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide). We validated that lipophilic fluorescence probe has a specific interaction with LDs and optimized staining conditions with each probes via systematic variations of physicochemical conditions. A protocol for quantitative measurement of accumulation kinetics of LDs in Chlamydomonas reinhardtii was developed using spectrofluorimeter and the accuracy of LD size measurement was confirmed by transmission electronmicroscopy (TEM). Second, unlike mammalian cells, investigation of microalgae in microfluidic system has been limited due to their small size and motility. Here, we present a simple surface immobilization method using gelatin coating as the biological glue. We have continuously monitored single microalgal cells for up to 6 days. Surface immobilization allowed high-resolution, live-cell imaging of mitotic process in real time-which followed previously reported stages in mitosis and LDs accumulation of suspension cultured cells. Third, PDMS (polydimethylsiloxane), which is the main component of microfluidic system, is hard to observe and manipulate cellular behavior with adsorption of hydrophobic fluorescence probes. Here, we present a new simple method for preventing unwanted hydrophobic absorption on PDMS device using Teflon coating. Throughout the clearing considered issues, we suggested a novel paradigm, never discussed, which could guarantee the most promising method for achieving economics of biodiesel. Based on miniaturized continuous culture system, we could generate various combinations of carbon and nitrogen source for measuring single cell behavior. This result indicate that single cell behavior under continuous culture system did not show similar result of conventional flask culture system. Throughput the result, cell size were affected by nitrogen concentration as well as intracellular lipid content were maximized by half deprivation of nitrogen. Especially, in mass (or continuous) culture system, it would be better to reduce only half of nitrogen source by considering economics of biodiesel. We hope that the applications of developed microfluidic platform become a useful tool for biodiesel research as well as system biology by helping high-throughput screening and biological relevant stimulus. This thesis also describes a new method for enhancing microalgal growth and intracellular lipid accumulation using vibration. As we previously mentioned, conventional microaglal biodiesel researches are only focused on the nutrient starvation that activates biosynthesis of lipid formation. However, these approaches slow down microalgal growth and development. Although these unfavorable conditions lead high lipid productivity at single cell level, their low growth rate are a major bottleneck considering commercial biodiesel production in the point of view at whole culture system. Due to this reason, we represent new concept for improving economics of biodiesel by realizing high growth rate and lipid productivity. Chlamydomonas reinhardtii were treated in the presence of various frequencies under mechanical vibrations and sounds. Mechanical vibration enhanced proliferation and we assumed that the cell displacement is the crucial factor for maximizing growth and development. The most interest thing is mechanical vibration boosts proliferation via enhanced carbon conversion ability. Although mechanical vibration system cause stress on photosynthetic system, all frequencies enhances growth yield. In cases of sound, except for 1000 Hz, all frequencies boosts proliferation as well as lipid production. Our results presented here confirmed that vibration can be a new method not only for boosting intracellular lipid formation but also for enhancing cell proliferation in normal condition. We hope that these properties could be an applicable condition for algal cultivation during biodiesel production.