1. Introduction
The bioreactor is a device when defined process conditions are provided for production in the form of cell suspension cultures, immobilized cultures, and organ and tissue cultures (Eibl & Eibl, 2008; Paz-Maldonado & González-Ramírez, 2014). Different types of bioreactors have been utilized in hairy root culture, liquid-phase, gas-phase, or hybrid reactors (Kim et al., 2002). The configuration of bioreactor significantly affects cultivation results by how controlling the optimum environment (Mishra & Ranjan, 2008).
Hyper-hydration is one of the most important disadvantages of liquid-phase in vitro culture, which is affected by light, CO2, and type of nutrient delivery (Correl et al., 2001; Towler et al., 2008). This leading to incomplete growth of the plant and finally the death of the plant in the ex-vitro condition (Ziv, 2000). Using a mist bioreactor can reduce the amount of hyper-hydration significantly (Correl et al., 2001; Chatterjeeet al., 1997). Mist bioreactor as a gas phase bioreactor exposed the surface of plant tissue directly to the nutrient mist and the samples are fed by gas-phase liquid medium (Perry & Green, 1997 ; Weathers et al., 1999). The Earlier design of the bioreactor was based on a nozzle to spread droplets of media with a diameter of 10-10000 µm (Weathers et al., 2008; Towler et al., 2008). In recent years bioreactors used an ultrasonic transducer submerged in the media for the production of mist. Utilization of mist bioreactor has grown steadily, due to its advantages where nutrient particle size reduced to (0.01-10 µm) thus the mass transfer improved in this case over liquid-phase reactors and allow effective gas exchange in densely growing biomass. Differing design of mist bioreactor dictated its application (Towler et al., 2008). The efficiency of three different configurations of nutrient mist bioreactor, inner loop nutrient mist bioreactor and modified inner-loop nutrient mist bioreactor on hairy root growth ofArtemisia annua was studied by Liu et al., (1999). The maximum and minimum growth index (final/initial dry weight) was 68 and 42 for modified inner-loop nutrient mist bioreactor and nutrient mist bioreactor, respectively (Liu et al., 1999). Furthermore, a comparative study of the effectiveness of mist and bubble column bioreactors on the production of artemisinin was done by Kim et al., (2001). Artemisinin content of roots grown in nutrient mist bioreactor, 2.64 mg/g DM, was nearly three times as much as roots grown in a bubble column, 0.98 mg/g DM (Kim et al., 2001).
Different operational conditions such as mist duty cycle, medium formulation, gas composition, and airflow rate also caused different results on physical properties (production thickness, color necrosis, etc.) (Wyslouzil et al., 2000; Dilorio et al., 1992). Among them, misting cycles have a direct impact on the amounts of nutrients, gas exchange, and oxygen available to plants, so that changes in misting cycles can have a significant effect on the quality of production. A cycle of 5 min of misting followed by 6 min of resting time was optimal for the growth of both safflower and beet hairy roots. A time longer than 5 min reduced biomass production while reduction of resting cycle to less than 5 did not have a significant effect on the growth of beet hairy root tissue. (Wyslouzil et al., 2000).
Three different methods, spinning disks, spray nozzles and ultrasound, are utilized to generate droplets in mist bioreactors (Weathers et al., 2008). The ultrasonic produced droplets are small and have a uniform size distribution (Weathers & Zobel, 1992). The ultrasonic atomization mechanism involves breaking up the thin film of liquid into fine droplets through the exposure of liquid to a high frequency (\(f>20\ \text{kHz}\)) vibrating surface (Sindayihebura & Bolle, 1998; Wood & Loomis, 1927). Two hypotheses of capillary wave and cavitation have been proposed by researchers to explain the ultrasonic atomization mechanism (Söllner, 1936; Peskin & Raco, 1963; Rooney, 1981). Boguslavskii and Eknadiosyants (1969) combined the capillary wave and cavitation theories and presented a ‘conjunction theory’. They expressed that interacts between the finite amplitude capillary waves and the periodic hydraulic shocks from the cavitation disturbance stimulate liquid to form droplets (Boguslavskii & Eknadiosyants, 1969). Since the liquid film is sonicated under high frequency and energy transducer the physical and chemical properties of liquid are prone to change. The transducer amplitude and frequency, liquid-film thickness, surface tension and density of liquid are the main parameters that affect droplet size distribution (Sindayihebura & Bolle, 1998). Therefore, a close study on the effects of expressed parameters and sonication on mist generating performance and physicochemical properties of media is necessary. The objective of this work is to study the change of physicochemical properties of different media under ultrasonic treatment and evaluate the effect of the liquid altitudes on the misting rate.