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.