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Parallel Computing and State Machine Software

Development for Multiple Temporary Immersion
Bioreactor Systems Sequ....

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 Parallel Computing and State Machine Software Development for
Multiple Temporary Immersion Bioreactor Systems Sequence Control

Poonpat Poonnoy1* and Nopmanee Topoonyanont2

1
Food Engineering Program, Faculty of Engineering and Agro-Industry, Maejo University,
Chiang Mai, 50290, THAILAND
2
Biotechnology Program, Faculty of Science, Maejo University,
Chiang Mai, 50290, THAILAND
*
Corresponding author: poonpat@mju.ac.th, ppoonnoy@gmail.com

Abstract
Temporary Immersion Bioreactor (TIB) system has been proven to be an efficient tool for
important economic crops micropropagation. This is mainly because the TIB system is capable
of improving plant quality and multiplication rates for ease of scaling-up while requiring low
production costs. A regular TIB system comprises two separate chambers: one for nutrient
medium storage and the other for culturing shoots. Liquid medium is transferred to the culture
tank and remains there for minutes, after which it is pumped back to the storage tank for reuse.
A control system plays a significant role in manipulating the flow of the liquid medium in the TIB
system. A simple control system consists of two digital timers working in parallel, responsible for
the operation of electromagnetic valves, which creates pressure difference between a liquid
medium and a culturing shoots chamber. Manual programming of the digital timers is a labor-
intensive task. The programming task becomes more critical especially when operating multiple
TIB systems. Incorrect programming of the digital timers may cause serious damage to the plant
production process, and devices and equipment connected to the TIB systems including the
operators working at the production site when there is insufficient pressure or overpressure. The
objective of this research was to develop the computer software to control the sequence of
multiple TIB Systems. Computer software was developed under LABVIEW environment based
on parallel computing and state machine architecture. The software consists of three
independent modules: 1) graphic user interface (GUI) module, 2) sequence control (SC) module,
and 3) hardware interface (HI) module. The GUI module received the information regarding
immersion time and immersion duration for each TIB system from the operator. The information
was automatically synchronized with the SC module, which initiated the immersion process at
the specified time and terminated the process after the immersion duration met the target. The
HI module continuously monitored the TIB sequence, assigned by the SC module, and triggered
the electromagnetic valves manipulating the air stream in the TIB system to force liquid medium
transport. The developed computer software was connected to eight 20-liter TIB systems and
tested under sugarcane plant micropropagation. It was observed that the computer software
could independently control the sequence of each TIB system in accordance with the specified
conditions. It requires less than one-minute changing immersion schedule and immersion time
without process interruption. The developed software can provide a convenient and effective
way to operate multiple 20-liter TIB systems with safety. It is possible to apply the software to
other similar TIB systems.
Keywords: Computerized Control System/Micropropagation/Parallel Computing/State Machine/
Temporary Immersion Bioreactor system
1. Introduction
Temporary immersion bioreactor has been considered as an efficient tool for plant
micropropagation. The temporary immersion system combines the advantages of the liquid and
solid medium culture by alternating the exposure of explants to nutrient liquid and air (Etienne
and Berthouly, 2002). The system allows the automated operation, which significantly reduces
the labor cost (Etienne and Berthouly, 2002). The temporary immersion system was successfully
used in micropropagation of numerous plant species such as sugarcane (Lorenzo et al., 1998),
and pineapple (Escalona et al., 1999). The achievement of the system is dependent upon many
factors including, but not limited to, the liquid medium volume, culture container volume,
immersion time, aeration, and forced ventilation (Dutta Gupta et al., 2006; Etienne and
Berthouly, 2002). The optimum condition for each species is different and varies in culturing
stage.
A regular TIB system comprises two separate chambers: one for nutrient medium storage and
the other for culturing shoots. Liquid medium is transferred to the culture tank and remains there
for minutes, after which it is pumped back to the storage tank for reuse. Transferring of the liquid
medium was controlled by a set of controller and actuator. For a simple pneumatic-driven
bioreactor system, two programmable digital timers are responsible for activation of two
electromagnetic valves, which supply compressed air to the TIB. The first valve is connected to
the medium storage tank; the other is connected to the culturing tank. The valve supplies
compressed air to the connected tank when it is energized. The valve releases the air when it is
not powered. Therefore, the liquid medium transfer between the culturing and the storage tank
can be made and controlled by manipulating the operation of the electromagnetic valves.
In order to fill the liquid medium into the culturing tank, positive pressure must be created inside
the storage tank. The liquid medium is transferred to the culturing tank immediately after the
pressure inside the storage tank overcomes the pressure drop due to frictions and liquid
elevation. At this stage, the electromagnetic valve connected to the storage tank is activated
while another valve connected to the culturing tank is not powered on. In other words, the
compressed air is supplied to the storage tank; the liquid medium is pressurized and transferred
to the culturing tank due to pressure difference. The air inside the culturing tank is displaced by
the liquid medium and escapes through the connected electromagnetic valve. In contrast, the
valves in opposed position are engaged when the liquid medium is drawn back to the storage
tank and when the force ventilation is needed. All electromagnetic valves are off during standby
and immersing stage; there is no liquid transfer at these stages.
The control signals are generated by the programmable digital timers. A synchronous schedule
for both digital timers must be precisely created and manually assigned to the digital timers. A
properly scheduled assignment allows the TIB to operate as expected; however, failure to do so
may cause serious damage to the plantlets, equipments and the operators. For example, both
electromagnetic valves are accidently activated; the compressed air is supplied to the TIB
resulting in excessive pressure. Depending on the pressure and material, the swelling of the
tanks may be observed. The disruption of the containers may be occurred if the exerted force is
higher than the ultimate strength of the material. Another example is the failure of the digital
timer responsible for the operation of the electromagnetic valve connected to the culturing tank.
The liquid medium will not be returned to the storage tank if the electromagnetic valve is not
activated as scheduled, leading to hyperhydricity and reduced productivity.
Scheduling the digital timers is a considerably complicated and time-consuming task. It becomes
critical when multiple TIBs are simultaneously engaged. Improper programming of the digital
timers due to human error may occur and cause serious damage to culturing shoots and
equipments, and injury to operators. Decent software allows the operator to configure the
temporary immersion systems in safety with less effort. The computer software for temporary
immersion system control may be developed based on a finite-state machine concept. This
paper presents the development of parallel state-machine computer software for multiple
temporary bioreactors operation.
2. Materials and method
2.1 Defining Temporary Immersion System Operational States
A pneumatic-driven temporary immersion system working principle was studied and modeled for
state-machine software development. The system consisted of two 20-liter transparent plastic
bottles (Nalgene, USA). Each bottle was covered with a plastic lid with three opening ports. The
ports were used for liquid medium and air transfers. Four normally closed one-way
electromagnetic valves were employed in airflow control. Utilizing four independent valves
allowed the culturing tank to be ventilated without air flowing through the liquid medium. The
water losses from the liquid medium due to evaporation were then reduced. A schematic
diagram of the system is shown in Figure 1.

Figure 1 Schematic diagram of the 20-liter temporary immersion system

The TIB system was operated in a specific sequence. The operation of the temporary immersion
system was time-dependent. A state-machine diagram of the TIB system was created as shown
in Figure 2. At the beginning, the TIB system was in standby mode where there was no fluid
transfer in the system. The computerized control system determined the on-off status of the TIB
set by the operator and checked for the immersion schedule if the bioreactor was on; otherwise,
it terminated the bioreactor operation. The control system initialized the filling liquid medium
process when reaching the time set. The liquid medium was filled into the culturing tank by
supplying compressed air to the storage tank; in the meantime, the displaced air was released
through the vent on the culturing tank. The control system calculated the filling process time (s)
by dividing the amount of liquid medium (liter) by the volumetric flow rate (liter/s). The bioreactor
turned into immersion state as the filling process stopped. During the immersion process, all
connected valves were closed, there was no fluid transportation until the immersion time was
reached. The immersion time was given by the operator. Similar to the filling process, the
draining process time was determined by dividing liquid medium volume by the volumetric flow
rate. Using liquid level sensor to control the filling and draining processes is possible but it may
lead to microbiological contamination (Simonton et al., 1991).
The control system checked the purge schedule when all liquid medium was in the storage. The
supplied- and released- valves on the culturing tank were activated simultaneously during the
TIB was in purging state. The culturing tank was then flushed with fresh air.

Figure 2 State-machine diagram for a TIB system

The boolean values for the valves were assigned regarding the TIB operational state as shown
in Table 1. The electromagnetic valve was activated to open the air-transferring port when the
boolean was set to true.
2.2 Computer software development
The computer software for multiple temporary immersion control based on state-machine
principle was developed under Labview environment. The operator could set the immersion and
forced ventilation frequency of each TIB independently by clicking on the buttons with time-
indicated on the GUI front panel (Figure 3). Immersion and ventilation (purge) duration could be
specified by changing the numeric value on the GUI (figure not shown). The SC module initiated
the immersion or forced ventilation processes regarding the specified values. The TIBs state
was monitored by the HI module, which sent high/low signals to the targeted digital output
device in accordance with the boolean for valves. The high signal was sent out when the
boolean value was true; otherwise, the low signal was sent out.
Table 1 : TIB state and connected valve operation

Electromagnetic valve boolean value

State Storage tank Culturing tank
Supply Release Supply Release
Standby False False False False
Filling liquid medium True False False True
Immersing culturing shoots False False False False
Draining liquid medium False True True False
Forced ventilation (purging) False False True True

In order to control eight TIBs simultaneously, eight SC modules were arranged in parallel.
Numerous global variables were specifically created for each TIB and deployed to serve the
parallel operation of the GUI module, the SC modules and the HI module.

Figure 3 Schematic diagram of the 20-liter temporary immersion system

3. Implementation of the software in sugarcane plantlets micropropagation

The developed software was compiled and executed on a personal computer (Intel Quad Core,
2.8 GHz, 2 GB of Ram) operated under Windows7 (R) environment. The PC was connected to a
32-channel sourcing digital output device (National Instruments: NI-9476) capable of supplying
24VDC to activate 32 electromagnetic valves controlling of fluid transfers in eight TIBs.
The developed system had been used in sugarcane micropropagation during elongation and
rooting stages. The starting material was sugarcane shoots that had been cultured for two
weeks on semi-solid medium. Each of eight TIB sets was filled with eighty shoots transferred
from the semi-solid medium. Ten liters of MS medium supplemented with 30 g/L sucrose was
added to each TIB sets. The nutrient solution was provided to the sugarcane shoots for 15
minutes/8 times a day. The light with an intensity of 70-120 µmol.m-2.s-1 was provided to the
shoots for 14 h a day. The temperature was controlled at 25±2º C. The experiment was carried
out for approximately three weeks. It was observed that the new shoots were divided into a
minimum of 5,000 usable shoots for each TIB set. In other words, the developed TIB system
 was capable of producing sugarcane of 40,000 shoots per batch. Determination of the survival
rate for the shoots after the plant conditioning process for one month revealed high survival rate
more than 98%.
It was observed that using the developed computer software allowed the operator to modify the
immersion conditions with less effort when compared with using conventional digital timers. The
computerized control system was able to control the sequence of multiple TIBs as given.
However, it was observed that liquid medium transfer rate between the storage tank and the
culturing tank reduced when the liquid medium hose was obstructed with small plantlets or when
insufficient air pressure was supplied to the TIB. Therefore, the filling and draining process times
needed to be extended to complete the liquid medium transfer. Manual adjustment of the liquid
medium transfer rate was required. Further study on the application of fluid flow and pressure
measurements in the TIBs together with appropriate computer algorithm is needed to improve
the capability of the software to detect and solve the fault in liquid medium transfer. The fault
detection function of the software may prevent the damage of plantlets due to hyperhydricity
caused by unintentional extended immersion duration.
4. Conclusion
Parallel computer software based on state-machine concept was developed to control multiple
20-liter pneumatic-driven temporary immersion bioreactor systems. The software was able to
control temporary immersion bioreactor system independently. The developed software had
been employed in sugarcane micropropagation during elongation and rooting stage. The TIB
systems controlled by the computer software were capable of producing 40,000 sugarcane
shoots per batch with a survival rate of 98%. Modification of immersion conditions for each
temporary immersion system could be simply made on screen. Additional feature on detection of
liquid transfer interruption due to small plantlets clog and insufficient air pressure should be
developed to minimize the risk of hyperhydricity.
5. Acknowledgement
The authors would like to express their sincere appreciation to the National Research Council of
Thailand (NRCT) for funds supporting throughout the research.
6. References
Dutta Gupta, S., Ibaraki, Y., Afreen, F., 2006. Temporary Immersion Bioreactor, In: Hofman, M.,
Anné, J., Nedović, V., Willaert, R. (Eds.), Plant Tissue Culture Engineering. Springer
Netherlands, pp. 187-201.
Escalona, M., Lorenzo, J.C., González, B., Daquinta, M., González, J.L., Desjardins, Y., Borroto,
C.G., 1999. Pineapple (Ananas comosus L. Merr) micropropagation in temporary
immersion systems. Plant Cell Reports 18, 743-748.
Etienne, H., Berthouly, M., 2002. Temporary immersion systems in plant micropropagation. Plant
Cell, Tissue and Organ Culture 69, 215-231.
Lorenzo, J., González, B., Escalona, M., Teisson, C., Borroto, C., 1998. Sugarcane shoot
formation in an improved temporary immersion system. Plant Cell, Tissue and Organ
Culture 54, 197-200.
Simonton, W., Robacker, C., Krueger, S., 1991. A programable micropropagation apparatus
using cycled liquid medium. Plant Cell, Tissue and Organ Culture 27, 211-218.

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