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WeathersFinalHR-07-010.05.09.08.pdf

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Electronic Journal of Integrative Biosciences 3(1):29-37. 07 October 2008

Special Issue on Hairy Roots (A. Lorence and F. Medina-Bolivar, co-editors) © 2008 by Arkansas State University

Mist reactors: principles, comparison of various systems, and case studies

Pamela Weathers1,2, Chunzhao Liu1,3, Melissa Towler2, and Barbara Wyslouzil4

1 Arkansas State University, Jonesboro, AR; 2 Worcester Polytechnic Institute, Worcester, MA;

3 National Key Laboratory of Biochemical Engineering, Beijing, China; 4 The Ohio State University, Columbus, OH *Corresponding author; email: weathers@wpi.edu

Keywords: Artemisia annua, scale-up, gas-phase reactors ABSTRACT

Growing hairy roots in bioreactors has proven challenging. Here we summarize the recent work using a novel bioreactor, the mist reactor, for the culture of hairy roots from many plant species. Compared to most liquid-phase bioreactors, this gas- phase bioreactor offers a completely different environment for growing roots. Design, modeling, and some of the unique engineering aspects inherent to mist reactors are explained along with some of the biological responses of roots when grown in mists compared to liquids. Prospects for future development of this technology are also summarized.

INTRODUCTION

Hairy roots can produce novel compounds not found in the whole plant or in untransformed roots (Flores and Medina- Bolivar, 1995; Wysokinska and Chmiel, 1997; Flores et al., 1998), often at levels exceeding those found in the parent plant (Wysokinska and Chmiel, 1997; Wysokinska and Rozga, 1998; Rao and Ravishankar, 2002). They also offer significant advantages for the production of engineered proteins (Guillon et al., 2006; Zhang et al., 2005; Shanks and Morgan, 1999). Although there are some reports of co-cultured differentiated tissues (e.g. shoots plus roots) being used to produce secondary metabolites (Mahagamasekera and Doran, 1998; Subroto et al., 1996), most efforts are focused on hairy roots.

Several bottlenecks, however, hamper the routine production of compounds, especially secondary metabolites, from hairy roots. The two main challenges are: (1) for many products the biochemical pathways and their regulation are not understood; and (2) although progress has been made, cost effective scaleable production systems are not yet well developed. Developing an inexpensive scaleable reactor for hairy roots would address the latter problem, enabling large- scale culture and the production of many important and valuable chemicals (Guillon et al., 2006). Recent political challenges to the release of transgenic plants further warrant the use of controlled production schemes including the use of low cost bioreactors (Guillon et al., 2006).

Our research has focused on gas-phase (mist) reactors, because this environment reduces the gas-exchange limitations and the high shear conditions normally found in

liquid-phase reactors. Unlike growth in liquid systems, roots grown in mist reactors are not oxygen limited (Weathers et al., 1999) even at high bed densities (Kim, 2001), and the production of secondary metabolites is often greater in mist reactors than in liquid phase reactors (Kim et al., 2001; Bais et al., 2002). High biomass density is required for a reactor system to be economically viable, and the maximum root tissue concentration that can be achieved in a bioreactor depends on the efficient mass transfer of oxygen and other nutrients into the dense root matrix (Curtis, 2000). A point in favor of gas phase reactors is that roots are more able to compensate for poor liquid dispersion than for poor gas dispersion within a reactor (McKelvey et al., 1993). A recent comparison by Suresh et al. (2005), also verified that a mist bioreactor is superior to other reactor designs for hairy root growth. Finally, establishing a hairy root culture on solid medium does not guarantee that they will grow well in liquid medium (Hallard et al., 1997), so gas-phase reactors may be the only option. Together these results provide compelling reasons to study mist reactors.

Recent reviews have added new perspectives regarding the prospects and challenges of producing secondary metabolites from hairy roots in bioreactors (Kim et al., 2002; Towler et al., 2006; Weathers et al., 2006). These reactors, however, have not yet been scaled to commercially useful sizes. The key scale-up concerns for hairy root bioreactors include the following: the flow and geometric configurations should be scale independent; high growth rates must be maintained with no limitations in liquid and gas (O2) nutrients; the key operating parameters must be identifiable and measurable so that scale-up can be achieved easily and economically (Cuello et al., 2003). Here we provide a review on mist/spray reactors and the efforts to scale them up.

LIQUID VS. GAS PHASE REACTORS

O2, CO2 and C2H4 are the three gases most important to plants, but all are poorly soluble in water; their solubility decreases with increasing nutrient concentration and temperature (Atkinson and Mavituna, 1991; Geankopolis, 1993), and oxygen is about 25 times less soluble than CO2. To ensure rapid growth of roots, high levels of oxygen are required while excessive CO2 accumulation must be prevented as the level of respiration increases with increasing root biomass. Liquids therefore limit the amount of gas available to growing roots.

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