development of multiplexed microcontroller...

DEVELOPMENT OF MULTIPLEXED MICROCONTROLLER BASED SYSTEM

FOR MICROBIOREACTOR

AHMAD YASER AL-HADDAD

A project report submitted in partial fulfillment of the

requirements for the award of the degree of

Master of Engineering (Electrical- Mechatronics & Automatic Control)

Faculty of Electrical Engineering

Universiti Teknologi Malaysia

JANUARY 2013

iii

I dedicate this to the revolts of Syria, to my parents, who have been supportive

throughout my life, to my grandparents, to my sisters, to my brothers, to all my

teachers who have inspired me a lot.

iv

ACKNOWLEDGEMENT

First and foremost, I would like to thank the almighty ALLAH (SWT) for

giving me the strength in doing and completing this project.

My sincere appreciation also goes to my supervisors Dr. Abdul Rashid Husain

and Dr. Muhd Nazrul Hisham for their continued guidance, support and

encouragement to ensure this work is a success. My earnest appreciation also goes to

all my colleagues for helping me in achieving this project.

I shall forever be grateful to my family for their belief in me and for their

unending support, financially; morally, spiritually and emotionally. Finally I would

like to express my sincere gratitude to my sister Hadia for her assistance and support.

v

ABSTRACT

Bioreactor is a device that widely used in fermentation process. Bench scale

bioreactor and shake flask are the most used devices to optimize such process. Due to

their high running cost and the high amount of labour required, new and better

alternative way must be found to carry on the optimization process. In the most recent

years, scientists have been working on smaller bioreactor of volume less than 1

millilitre and became known as microbioreactor. This device and due to its small

volume size should bring more benefits and potential to optimization process in

fermentation. Since microbioreactor stills in development phase, researches till date

are trying to come with ideas and designs to be adopted later as acceptable industry

standard. In the literature, there have been many designs attempts to create a platform

to support one or more microbioreactors. Many of these attempts have achieved great

results with comparable performance to that of bench scale bioreactor. Most the

previous designs are made with the usage of data acquisition card from National

instrument. The trend toward NI DAQ is due to the simplicity in programming

environment, being user friendly and many other features. Biochemical engineers

could settle for alternative and cheaper way, if they are willing to give up some of the

convenient and features provided by NI DAQ. In this project, this alternative method

is being investigated, tested and used. The goal is to use microcontroller as DAQ to

provide controlling, data logging and monitoring of temperature and speed control for

microbioreactor. This setup will be totally microcontroller based and using dedicated

electronic circuit to achieve the objectives. The results for temperature and agitation

control that have been obtained are satisfactory and now this setup is ready to be used

on actual microbioreactor.

vi

ABSTRAK

Bioreaktor adalah alat yang digunakan secara meluas dalam proses penapaian.

Bioreaktor skala jenis duduk dan kelalang digunakan secara meluas bagi tujuan

pengoptimuman. Disebabkan kos pengoperasian dan jumlah tenaga kerja yang ramai

diperlukan, kaedah alternative yang baru dan lebih baik diperlukan. Sejak beberapa

tahun kebelakangan ini, saintis telah meneruskan kajian mereka menggunakan

isipadu kurang dari 1 milimeter dan dikenali sebagai microbioreaktor. Peranti ini dan

disebabkan oleh julat kerja yang kecil, ia sepatutnya memberikan kelebihan dan

keupayaan tinggi untuk proses pengoptimuman dalam proses penapaian.

Memandangkan proses mikrobioreaktor masih dalam fasa pembangunan, para

penyelidik masih mengkaji idea-idea dan rekabentuk-rekabentuk yang baru untuk

diadoptasi kemudiannya agar selari dengan standard industri. Dalam kajian yang

dilakukan, terdapat banyak kajian rekabentuk dilakukan bagi tujuan untuk membina

satu platform untuk menyokong satu atau lebih mikrobioreaktor. Kebanyakkan hasil

kajian ini telah mencapai keputusan yang memberangsangkan jika dibandingkan

dengan bioreactor skala jenis duduk. Kebanyakkan rekabentuk-rekabentuk asal dibina

dengan menggunakan kad perolehan data (DAQ) dari National Semiconductor (NI).

Trend masa kini ke arah penggunaan kad DAQ adalah disebabkan oleh mudahnya

pengaturcaraan, mudah diguna, dan ciri-ciri yang lain-lain. Jurutera biokimia

memiliki alternatif dan kaedah yang murah, sekiranya mereka sanggup

mengorbankan kemudahan dan ciri-ciri yang disediakan oleh NI DAQ. Dalam projek

ini, kaedah alternatif ini dikaji, diuji, dan digunakan. Matlamatnya adalah untuk

menggunakan mikropengawal sebagai DAQ untuk membekalkan kawalan, perekodan

data, dan pemantauan suhu dan kawalan kelajuan untuk mikrobioreaktor. Persediaan

ini adalah berdasarkan mikropengawal sepenuhnya dan menggunakan litar elektronik

untuk mencapai objektifnya. Keputusan bagi kawalan suhu dan pergolakan yang

diperolehi adalah memuaskan dan persediakan ini adalah siap dan boleh digunapakai.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES x

LIST OF FIGURES xi

LIST OF ABBREVIATIONS xiv

LIST OF SYMBOLS xv

1 INTRODUCTION

1.1 Introduction 1

1.2 Problem Statement 3

1.3 Project Objectives 5

1.4 Scope of the Project 5

1.5 Summary of the Chapter 6

2 LITERATURE REVIEW

2.1 Devices used in microbial cultivation 7

2.1.1 Bench scale Bioreactor 7

2.1.2 Shake flask culture 9

2.1.3 Microbioreactor 12

2.1.4 Comparison 14

viii

2.2 Considerations in microbioreactor design 17

2.2.1 Fabrication materials 17

2.2.2 Mixing 18

2.2.3 Temperature 19

2.2.4 pH 21

2.2.5 Evaporation 22

2.3 Previous works on microbioreactor 23

2.3.1 Development of a single-use microbioreactor by

D. Schäpper el. al (2010) 23

2.3.2 Development of a multiplexed microbioreactor

system by Szita N el. al (2005) 28


3 DESIGN AND DEVELOPMENT

3.1 Introduction 34

3.2 Hardware development 35

3.2.1 Temperature Measurement 35

3.2.1.1 The LM35 35

3.2.1.2 The PT100 37

3.2.2 Speed Measurement 46

3.2.3 Actuators 52

3.2.3.1 Motor 52

3.2.3.2 Heater 53

3.2.4 DAQ 55

3.3 Software Development 56

3.3.1 Arduino Software 56

3.3.2 GUI 57

3.3.2.1 Setting up the interface 58

3.3.2.2 Extra Considerations 60

3.3.2.3 Simple Example 64

3.3.3 PI Controller 65

3.3.4 PCB design 66


ix

4 RESULTS AND DISCUSSION

4.1 Introduction 69

4.2 First phase 70

4.2.1 Hardware 70

4.2.2 Temperature control 71

4.2.3 Speed control 73

4.3 Second phase 75

4.3.1 Hardware 75

4.3.2 Temperature control 77

4.3.3 Speed control 78

4.4 LabView GUI 82


5 CONCLUSION AND FUTURE PLAN

5.1 Conclusion 88

5.2 Future plan 89

REFERENCES 90

Appendix A 93

x

LIST OF TABLES

TABLE NO. TITLE PAGE

2.1 Comparison 15

3.1 PT100 Chart of values 38

3.2 PI parameters 65

4.1 Testing PI parameters 79

xi

LIST OF FIGURES

FIGURE NO TITLE PAGE

1.1 New Brunswick bioreactor 3

1.2 Prototype microbioreactor with 100 μl working volume 3

2.1 Bench scale bioreactor 9

2.2 Shake flask 10

2.3 Shake flask shaker 10

2.4 Shake flask culture 11

2.5 Controlled environment chamber 12

2.6 Microbioreactor platform at UTM 13

2.7 The microbioreactor mechanical design 13

2.8 Mixing scheme through the mounting of stirrer bar on a revolting

central shaft 19

2.9 The PT100 Temperature sensor 20

2.10 Photo of the complete microbioreactor seen from the bottom 24

2.11 Sketch of both main reactor layers as seen from below 24

2.12 Mixing in the microbioreactor at 500rpm 26

2.13 Temperature response at different set points 27

2.14 Displaying set points and the measured values of pH 28

2.15 The reactor design 29

2.16 Photograph of the multiplexed microbioreactor system 29

2.17a Parallel fermentations of E. coli 31

2.17b Parallel fermentations of E. coli 31

2.18 Comparison with bench scale bioreactor 32

3.1 LM35 terminal connections 35

3.2 Circuit schematic of the LM35 followed by the amplifier 36

xii

3.3 PT100 37

3.4 Transmitter 39

3.5 Wheatstone bridge 39

3.6 Difference amplifier 41

3.7 Non-inverting operational amplifier 42

3.8 Low-Pass filter 43

3.9 Conditioning circuit for the PT00 44

3.10 Simulation of PT100 circuit in Protus 45

3.11 Hall-effect sensor 46

3.12 Terminal connections 46

3.13 ATMEGA328P Pins mapping 47

3.14 Complete Circuitry for speed measurement 48

3.15 Flow chart for speed measurement 50

3.16 Stages of development 53

3.17 Control circuit 54

3.18 Arduino mega 2560 55

3.19 Arduino IDE 56

3.20 Firmware for Arduino 59

3.21 Arduino Functions in LabView 59

3.22 Init VI 61

3.23 Init Front Panel 61

3.24 Init Block Diagram 62

3.25 Example 64

3.26 First Design 66

3.27a Second design main board 67

3.27b Expansion to the second design 68

4.1 Hardware of the first stage 70

4.2 Temperature control response 71

4.3 Testing condition where the heater placed underneath 71


4.5 Testing condition where the heaters placed on the side 73

4.6 Speed control response 74

4.7 Main board 75

4.8 Optional expansion 76

xiii


4.10 Testing condition where the heater placed underneath 77

4.11 Speed control response 78

4.12 Testing condition 78

4.13 Speed control response for set one 79

4.14 Speed control response for set two 80

4.15 Mixing Quality test at 300rpm 81

4.16 Block diagram for temperature control 82

4.17 Front Panel for temperature control 83

4.18 Block diagram for speed control 84

4.19 Front Panel for speed control 85

4.20 Block diagram of the combined GUI 86

4.21 Front panel of the combined GUI 86

4.22 Testing conditions 87

xiv

LIST OF ABBREVIATIONS

DAQ - Data Acquisition Card

DO - Dissolved Oxygen

OD - Optical Density

NI - National Instruments

PID - proportional-integral-derivative

GUI - Graphical User Interface

rpm - revolution per minute

PMMA - polymethylmethacrylate

PDMS - polydimethylsiloxane

RTD - resistance temperature detectors

S/V - surface area to working volume ratio

ISFET - ion-sensitive and field-effect transistor

PWM - Pulse-width modulation

ADC - Analog Digital converter

PCB - printed circuit board

IC - integrated circuit

xv

LIST OF SYMBOLS

mL - millilitre

µL - microlitre

°C - celsius

V - voltage/volts

mV - millivolt

µV - microvolt

A - ampere

mA - milliampere

Ω - ohms

µF - microfarad

Hz - hertz

CHAPTER 1

INTRODUCTION

1.1 Introduction to Bioreactors

A bioreactor as shown in Figure 1.1 is simply a vessel usually cylindrical in

shape that used to carry out fermentation experiments. A bioreactor normally integrated

with tight on line measurements and process control capabilities. These features are

critically important such that fermentation experiments in bioreactor can be performed

under controlled conditions as stated by Bailey el. al (1986). For a standard bioreactor

system, it is norm to have a feedback control loop over physical parameters such as pH,

temperature, agitation, dissolved oxygen concentration and water level.

Fermentation is a process that is widely performed in many sectors such as

biotechnology, food processing, health care, agriculture and environmental management

as stated by Szita N el. al (2005). They are many devices that are in use to optimize such

processes for example bench scale bioreactors or shake flasks. These devices range in

terms of the volume size and capabilities. Bench scale biroeactors normally operated

between working volume of 1 liter and 10 liters where else shake flasks utilized much

lower volume i.e. less than 2 liters. In relative to bench scale bioreactors, shake flasks

2

usually do not integrated with sensors and actuators. Mixing is based on shaking

principle and operation is generally limited to batch mode operation only.

In the most recent years, scientists have been attempting to create smaller

bioreactor design that comprises the advantages of both types of experimental tools

(shake flasks and bioreactor) in one small unit named as microbioreactor shown in

Figure 1.2. The drastic reduction in volume’s size to less than 1mL will reduce the cost

dramatically and open the possibilities of running multi microbioreactors simultaneously

(multiplexing) hence increasing the throughput. As stated by D. Schäpper el. al (2009)

microbioreactor would not only reduce the fabrication cost per bioreactor compared to

bench scale, but will also add more advantages such as reduced running cost through the

reduction of substrate required, reduced power requirements, reduced space

requirements especially in parallel operations and finally reducing the effort and labour

required to prepare the bioreactor through the usage of disposable microbioreactors.

Even though dramatic cost reduction can be obtained using microbioreactors as

previously mentioned, another part of the microbioreactor platform circuit design still

suffering from high cost and still need to be considered. The circuit design includes all

the necessary electrical components that are needed to interface and control the

microbioreactor such as Data Acquisition Card (DAQ), temperature sensor transmitter

and any possible actuators. Most of the work previously performed on microbioreactor

such that by Szita N el. al (2005), M.N.H.Z Alam el. al (2010), D. Schäpper el. al

(2010) and Lee HL el. al (2006) are all made using high end DAQ from National

instrument. The trend toward NI DAQ is due to many features provided and the

convenient when it comes to programming, but that comes with a price. Since many of

the great capabilities and features of NI DAQ are being paid for and not being used,

alternative and cheaper way could be used as replacement if giving up some of the

convenience provided by NI DAQ is not an issue.

3

The aim of this project is to present cheaper and effective way with comparable

results to replace the expensive and bulky circuitry that is usually used on

microbioreactor design. Many of the devices around us are being incorporated with low-

cost microcontroller into their circuitry design to perform many tasks effectively. Part of

this project is to introduce the microcontroller into micobioreactor circuit design to

replace the DAQ without dramatic lose in the performance. The microcontroller based

DAQ should handle data logging, controlling and monitoring.

1.2 Problem statement

On line measurements and process automation is a necessity for microbioreactor

operation. In microbioreactor operation, often sensors and actuators from the

microbioreactor system are connected to a data acquisition device for in- and output of

signal during a standard process control routine. The monitoring and process control

tasks in microbioreactor are normally complex as it involves more than one feedback

Figure 1.2 Prototype microbioreactor with

100 μl working volume developed in the

BIOS/Lab-on-a-Chip group

Figure 1.1 New Brunswick bioreactor

4

control looping. As reported by Szita N el. al (2005), data such as temperature, dissolved

oxygen, agitation, pH and optical density are typically the measured and controlled

variables in microbioreactor operation. Due to limited knowledge on computer

programming, microbioreactor operators (mostly Biochemical Engineers) would go for a

readily available data acquisition device to execute the above mentioned tasks. One of

the most widely utilized data acquisition devices is the DAQ produced from National

Instruments (NI). As example, NI USB 6343 consisting of 16 number of analog input, 4

number of analog output, and 48 number of digital input/output with 500 kS/s sampling

rate. Despite these distinctive features which obviously can be beneficial for process

automation of microbioreactor operation, there are relatively expensive compared to a

standard microcontroller platform.

Microcontroller based DAQ could be of great replacement to typical DAQ at

much lower cost, provided that some of the convenient and features must be given up.

For example, most of microbioreactor operations are relatively slow and take long time

to respond, thus paying extra money for DAQ to have a very high sampling time isn’t

necessary. Microbioreactor would work perfectly with low sampling time without the

risk of any loses in terms of the performance and the controlling ability. Any typical

microcontroller consists of many digital and analog pins. For example, Mega 2567

consists of 16 analog pins, and 15 PWM pins that can be used as analog outputs and 54

pins of digital input/output. When paired with LabView, Mega 2567 can achieve

sampling rate up to 125 S/s.

5

1.3 Project objectives

The main objective of this project is to design a low-cost microcontroller based

platform as data acquisition device for microbioreactor operations.

1.4 Scope of the project

The scope of this project is divided into three main categories. There are as follows:-

a) Microbioreactor design

(i) A simple polymer based microbioreactor design that is cylindrical in

shape will be used.

(ii) Working volume of the microbioreactor is 600microliter.

(iii) The microbioreactor should be integrated with suitable temperature and

agitation (via magnetic stirrer bar) features.

(iv) PT100 temperature sensor will be used for temperature measurement of

the microbioreactor content.

(v) A hall-effect magnetic sensor will be used to measure the rotational speed

of a DC motor used for the agitation control.

(vi) The performance of the temperature and the agitation control will be

evaluated based on the controller accuracy, response time, and settling

time through the set-point tracking experiments.

(vii) A Proportional-Integral-Derivative (PID) control algorithm will be

implemented for the temperature and the agitation control.

(viii) Data on the performance of the temperature and the agitation control of

the microbioreactor will be collected and discussed.

6

b) Microcontroller platform

i) Arduino based microcontroller will be used as data acquisition card and it

should handle the controlling, monitoring and data logging.

ii) Another dedicated microcontroller will be used mainly to handle the

speed measurement and to convert rpm measurements into voltage levels

to be read by the Arduino microcontroller

c) Programming for control routines and data logging

i) Programs for control routines and data logging of the microbioreactor

operation will be written by using the GUI LabVIEWTM

(National

Instruments, US) software.

ii) The programs will be implemented by interfacing the microcontroller

platform with the microbioreactor system.

1.5 Summary of the chapter

In this chapter, brief introduction to bioreactor and microbioreactor been

presented. Next, the problem that still exists with the previously proposed designs in the

literature been highlighted. Finally, the steps and measurements taken to overcome them

and they are all presented as scope of this project.

90

REFERENCES

1. Bailey, J. E. and Ollis, D. F. “Biochemical Engineering Fundamentals, ” 2nd edition.

McGraw-Hill, 1986

2. Szita. N., Boccazzi. P., Zhang. Z., Boyle. P., Sinskey. A.J. , Jensen. K.F. , “Development

of a multiplexed microbioreactor system for high-throughput bioprocessing,” Lab Chip,

2005, 5(8), pp. 819-826

3. D. Schäpper, M.N.H.Z. Alam, Szita. N, A. Eliasson Lantz, K. V. Gernaey , “Application

of microbioreactors in fermentation process development: a review” Springer-Verlag,

2009 Anal Bioanal Chem ,395, pp. 679–695

4. M.N.H.Z. Alam, D. Schäpper, K. V. Gernaey, “Embedded resistance wireasheating

element for temperature control in microbioreactors, “ J. Micromech. Microeng., 20,

2010, 055014 (10pp)

5. D. Schäpper, S. M. Stocks, Szita. N, A. Eliasson Lantz, K. V. Gernaey, “Development of

a single-use microbioreactor for cultivation of microorganisms,” Chemical Engineering

Journal, 2010, 160, pp. 891-898

6. Lee HLT, Boccazzi P, Ram RJ, Sinskey AJ, “Microbioreactor arrays with integrated

mixers and fluid injectors for high-throughput experimentation with pH and dissolved

oxygen control,” Lab Chip, 2006, 6, pp. 1229–1235

7. Zhao, Y. G., Lu, W. K., Ma, Y., Kim, S. S., Ho, S. T.,. Marks, T. J., Appl. Phys. Lett.

2000, 77, 2961–2963.

8. Fleger M, Neyer A “PDMS microfluidic chip with integrated waveguides for optical

detection, ” Microelectron Eng 83:1291–1293

9. Zhang Z, Boccazzi P, Choi HG, Perozziello G, Sinskey AJ, Jensen KF, “Microchemostat-

microbial continuous culture in a polymer-based, instrumented microbioreactor,“ Lab

Chip,2006, 6, pp. 906–913

91

10. Maharbiz MM, Holtz WJ, Howe RT, Keasling JD, “Microbioreactor arrays with

parametric control for high-throughput experimentation,” Biotechnol Bioeng , 2004,

85(4), pp. 376–381

11. Krommenhoek, E.E., Van Leeuwen, M., Gardeniers, H., Van Gulik, W.M., Van Den

Berg, A., Li, X., Ottens, M., Van Der Wielen, L.A.M., Heijnen, J.J., “Lab-scale

fermentation tests of microchip with integrated electrochemical sensors for pH,

temperature, dissolved oxygen and viable biomass concentration,” Biotechnology and

Bioengineering, 2008, 99 (4), pp. 884-892

12. Petronis S, Stangegaard M, Christensen CBV, Dufva M, “Transparent polymeric cell

culture chip with integrated temperature control and uniform media perfusion,”

Biotechniques, 2006, 40,pp. 368–376

13. Maiti TK , “A Novel Lead-Wire-Resistance Compensation Technique Using Two-Wire

Resistance Temperature Detector 2006, ” IEEE Sens J 6(6):1454–1458

14. Vervliet-Scheebaum M, Ritzenthaler R, Normann J, Wagner E, “Short-term effects of

benzalkonium chloride and atrazine on Elodea canadensis using a miniaturised

microbioreactor system for an online monitoring of physiologic parameters,” Ecotoxicol

Environ Saf , 2008, 69, pp. 254–262

15. Van Leeuwen, M., Heijnen, J.J., Gardeniers, H., Oudshoorn, A., Noorman, H., Visser, J.,

van der Wielen, L.A.M., van Gulik, W.M., “A system for accurate on-line measurement

of total gas consumption or production rates in microbioreactors” Chemical Engineering

Science, 2009, 64 (3), pp. 455-458

16. Zanzotto A, Szita N, Boccazzi P, Lessard P, Sinskey AJ, Jensen KF, “Membrane-aerated

microbioreactor for high-throughput bioprocessing,” Biotechnol Bioeng, 2004, 87(2), pp.

243–254

17. John GT, Goelling D, Klimant I, Schneider H, Heinzle E. “pH-Sensing 96-well microtitre

plates for the characterization of acid pro-duction by dairy starter cultures, ” J Dairy Res,

2003, 70(3):327–333.

92

18. Krommenhoek, E.E. and Gardeniers, J.G.E. and Bomer, J.G. and Li, X. and Ottens, M.

and Dedem van, G.W.K. and Leeuwen van, M. and Gulik van, W.M. and Wielen van der,

L.A.M. and Heijnen, J.J. and Berg van den, A. “Integrated electrochemical sensor array

for on-line monitoring of yeast fermentations, ” Analytical Chemistry, 2007, 79 (15). pp.

5567-5573

19. De Jong J “Application of membrane technology in microfluidic devices,” (Ph.D. thesis).

University of Twente, 2008

20. Wu M.H., Huang S.B, Cui Z.F, Cui Z., Lee G.B, “A high throughput perfusion-based

microbioreactor platform integrated with pneumatic micropumps for three-dimensional

cell culture. Biomed,” Microdevices 2008, 10, 309-319.

21. Buchenauer A, Hofmann MC, Funke M, Büchs J, Mokwa W, Schnakenberg U. “Micro-

bioreactors for fed-batch fermentations with integrated online monitoring and

microfluidic devices,” Biosens. Bioelectron, 2009, 24:1411 –1416.

22. Ali Aminian, Marian K. Kazimierczuk, “Electronic Devices: a design approach”, Pearson

Prentice Hall, 2004

23. Thomas L. Floyd, “Electronic Devices: Conventional Current Version”, 8th Ed., Pearson

Prentice Hall, 2008

24. Muhammad H. Rashid, “Microelectronic Circuits: Analysis and Design”, PWS

Publishing Company, 1999

25. Sergio Franco, “Design with Operational Amplifiers and Analog Integrated Circuits,”

McGraw-Hill, 2002/1988

26. J. Millman, C. C. Halkias, “Integrated Electronics: Analog and Digital Circuits and

Systems,” McGraw-Hill, 1971

development of multiplexed microcontroller...

Documents