Using Low Cost Components To Determine Chlorophyll

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Using Low Cost Components To Determine Chlorophyll

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Using Low Cost Components To Determine Chlorophyll Concentration By Measuring Fluorescence Intensity
Hannes Truter
Supervised by Dr Fred Nicolls Submitted to the Faculty of Engineering, University of Cape Town, in fulfilment of the requirements for the Degree of Master of Science

Acknowledgements
I would like to thank the following people for their contribution towards this thesis:
• Dr Fred Nicolls, my supervisor, for his willingness to accept me in his student group. His patience and guidance over a long period was very encouraging when some of the practical measurements and data did not look promising.
• Dr Clive Garcin for going out of his way to help me by providing me with algae and chlorophyll samples, without which I would have been unable to make any useful measurements during the development of the fluorometer.
• Dr Stewart Bernard for all the valuable time he set aside to provide me with information and guidance on chlorophyll fluorescence and the requirements of the low cost fluorometer.
• Trevor Probyn for allowing me to join them during their instrument calibration in a laboratory. He then also scanned the chlorophyll calibration standard to provide me with a wavelength profile which was invaluable to interpret the measurement data.
• Emma Bone for her help during the laboratory measurements of chlorophyll a fluorescence that was required to determine the chlorophyll a measurement range of the fluorometer system.
• Rick Collins of Arius Inc. (www.arius.com) for helping me to find a better digital signal processing method that could be used to recover the modulated fluorescence signal amidst noise.

Declaration
I, Johannes P. Truter, declare that this dissertation is my own work except where otherwise stated. It is being submitted for the degree of Master of Science in Engineering at the University of Cape Town. It has not been submitted before for any degree or examination at this or any other university.
Signature of Author:
April 2015 Cape Town

Abstract
This dissertation describes the development of a low cost fluorometer with the aim of using it as an algae and phytoplankton concentration sensor. As it forms the core of this fluorometer's functionality, chlorophyll's fluorescence characteristics and origins are discussed. Special attention is given to the variability of chlorophyll fluorescence as it has a big influence on measurements. Experimental procedures and data are provided to show why each component was finally selected for use in the fluorometer. An analogue front end device with programmable gain on each 24-bit ADC channel forms the interface between the high sensitivity TSL257 light-to-voltage light sensors and the 32-bit ARM microcontroller that controls the system. The microcontroller software controls the 470 nm LED current to create a 75 ms light pulse that has a 63 Hz sine wave modulated on it. The low cost light sensors proved to be sensitive enough to detect the low light intensities of chlorophyll fluorescence. The challenges of measuring the low level voltages from these light sensors are discussed. The amount of noise on the light sensor voltages at low chlorophyll concentrations make it difficult to accurately measure the fluorescence signal. Different light modulation and digital signal processing techniques were investigated to compare the effective recovery of the fluorescence signal. Sine wave modulation along with sample averaging provided good results. The results of laboratory experiments with pure chlorophyll α and extracted chlorophyll are discussed to give an overview of the capabilities and limitations of the developed fluorometer that is able to measure the fluorescent light from extracted chlorophyll concentrations as low as 0.01 µg/l.

Table of Contents
1 Introduction ............................................................................................................................... 1 1.1 The Thesis Project History ...........................................................................................1 1.2 The Concept Design .......................................................................................................3 1.3 Layout of This Thesis .....................................................................................................6
2 W hat is Chlorophyll Fluorescence? .................................................................................12 3 Chlorophyll Fluorescence Measurement ........................................................................15
3.1 The Kautsky or OJIP Curve .......................................................................................15 3.2 Chlorophyll Fluorescence Nomenclature and Measurement Analysis ............18 3.3 Chlorophyll Fluorescence Variabilit y .......................................................................25 3.4 Chlorophyll Concentration Measurement ...............................................................31 4 T he Excitation Light System ..............................................................................................34 4.1 Excitation Light Wavelengths ....................................................................................34 4.2 Excitation Light Sources .............................................................................................39 4.3 Controlling the Excitation Light Intensit y ...............................................................43 4.4 Modulating the Fluorescence Excitation Light ......................................................49 5 T he Fluorescent Light Measurement ...............................................................................52 5.1 Finding the Right Light Sensor .................................................................................53 5.2 Using Light Filters ........................................................................................................56 5.3 Light Sensor Location .................................................................................................61 5.4 Light Intensit y Changes for Different Chlorophyll Concentrations ..................63 5.5 Improving the Fluorescence Light Sensor Signal Qualit y ..................................66 5.6 Recovering the Fluorescence Signal .......................................................................67 6 T echnical Details of the FICC ...........................................................................................70 6.1 The mbed Development Board ..................................................................................70 6.2 LED Current Control ....................................................................................................71 6.3 Modulation of the Light Source .................................................................................73 6.4 The FICC Front End Assembly ..................................................................................82 6.5 The MCP3903 Analogue Front End ..........................................................................85 6.6 The LabVIEW User Interface and Data Storage ...................................................86 7 FICC Practical Evaluations ................................................................................................87 7.1 Measurements wit h a Chlorophyll Extract ..............................................................87
7.1.1 First Spinach Chlorophyll Extraction ...............................................................87 7.1.2 Second Chlorophyll Extraction ..........................................................................88 7.2 Measurements wit h a Calibration Standard ...........................................................93 7.2.1 Chlorophyll α Calibration Standard Preparation and Measurement .......93 7.2.2 Chlorophyll a Calibration Standard Measurement Results .......................96 7.3 FICC Calibration.........................................................................................................102 8 T hesis Conclusions ............................................................................................................104 8.1 Current Status of the FICC ......................................................................................104 8.2 Further Investigations for the FICC .......................................................................107 9 Appendix A........................................................................................................................... 111
Figure Index
Figure 1: FICC system diagram ..............................................................................................4 Figure 2: Chlorophyll energy levels from light absorption. ...........................................13 Figure 3: Kautsky / OJIP curve of a pea leaf. ..................................................................16 Figure 4: Fluorescence rise wit h and without DCMU. ....................................................22 Figure 5: Maximum fluorescence variations. ....................................................................26 Figure 6: Effect on fluorescence of Chlorella vulgaris sinking. ...................................29 Figure 7: Effect of stirring on fluorescence measurement. ...........................................30 Figure 8: Chlorophyll excitation and emission spectra. .................................................35 Figure 9: Chlorophyll α and b excitation wavelength responses. ...............................38

Figure 10: White LED emission spectrum. ........................................................................40 Figure 11: 1206 Size SMD LED fluorescence. .................................................................42 Figure 12: Cut LED & SMD LED layout. .............................................................................43 Figure 13: LED current vs LED light intensit y. .................................................................45 Figure 14: LED intensit y vs current relationship during sine modulation. ................46 Figure 15: LED wavelength shift for different forward currents. .................................47 Figure 16: Effect of LED wavelength shift. ........................................................................48 Figure 17: Modulated light pulses with different DC offsets. .......................................49 Figure 18: OPT 101 (10x gain) delay versus TSL250. ....................................................55 Figure 19: TSL250 & TSL257 comparison. ........................................................................56 Figure 20: Effect of different filters on measured fluorescence. .................................59 Figure 21: Fluorescence on opposite sides. .....................................................................62 Figure 22: Light sensors next to LED. ................................................................................63 Figure 23: Light intensit y versus concentration. ..............................................................64 Figure 24: Fluorescence intensit y for different concentrations. ..................................65 Figure 25: Effect of concentration and distance on light intensit y. ............................66 Figure 26: FICC system diagram. ........................................................................................70 Figure 27: LED current control circuit. ...............................................................................72 Figure 28: Current control accuracy ...................................................................................73 Figure 29: Constant light fluorescence curve. .................................................................74 Figure 30: Kautsky curve seen over different durations. ..............................................75 Figure 31: Modulated excitation light pulse. .....................................................................76 Figure 32: Maximum fluorescence for pulse width. .........................................................77 Figure 33: Light pulse duration effect. ...............................................................................78 Figure 34: Fluorescence rise over 75 ms. .........................................................................78 Figure 35: Fluorescence change with unmodulated 250 ms pulses. ..........................79 Figure 36: Fluorescence intensit y change for consecutive pulses. ............................80 Figure 37: Fluorescence change with 2 s sine wave modulation. ...............................81 Figure 38: 100 ms & 500 ms sine wave pulses effect. ...................................................81 Figure 39: Protot ype housing & layout. ..............................................................................83 Figure 40: FICC flat face protot ype. ...................................................................................84 Figure 41: LabVIEW GUI. .......................................................................................................86 Figure 42: Spinach extracted chlorophyll absorbance spectrum. ...............................88 Figure 43: Fluorescent to excitation light ratio. ...............................................................90 Figure 44: Excitation to fluorescence ratio for concentrations. ...................................91 Figure 45: Excitation light changes for concentration and current. ...........................92 Figure 46: Absorption spectrum of calibration standard. ..............................................96 Figure 47: Averaged fluorescent light intensities. ...........................................................98 Figure 48: Moving average fluorescence vs chlorophyll concentration. ...................99 Figure 49: Modulated sine wave DC offset change. .....................................................100 Figure 50: FFT "brick wall” filtered signals & unfiltered signals. ..............................100 Figure 51: “Brick wall” FFT filtered fluorescence relationship to concentration.. 101
Table 1: TSL250 & OPT101 comparison. ...........................................................................54

Chapter 1 Introduction
1 Introduction
This chapter starts with the background to why this thesis project was started before presenting the basic concept design of the system that was used for the laboratory experiments to confirm the system's functionality. The last section of the chapter introduces the layout of the rest of the thesis. It also provides some high level detail about the different topics that are discussed in each chapter.
1.1 The Thesis Project History
The idea for the project started when two research groups required a low cost sensor to measure algae concentrations in water. These groups were the Centre for Bioprocess Engineering Research (CeBER), at the University of Cape Town, and the Earth Observation group, at the Council for Scientific and Industrial Research (CSIR).
CeBER grows algae in the laboratories for research in different areas. These include production of oil for biodiesel as well as research into valuable pigments (phycocyanin, astaxanthin) and other biological products. In all these research areas it is necessary to know the algae concentration. Low cost algae concentration sensors would free up money for the main research topics.
The CSIR group required a low cost sensor to measure algae concentrations in dams in South Africa as well as phytoplankton concentrations in the ocean along South Africa's coast line. Algae in oceans form part of the phytoplankton group, which also contain other organisms, that makes use of photosynthesis for survival. Algae and phytoplankton have enough similar characteristics to make it possible to measure their concentrations in water with the same instruments. This is discussed in much more detail later in the thesis.
The CSIR group uses data from remote sensing equipment such as satellites. At the time they needed a sensor that could be fitted to a submersible float for several months to measure and log the algae or phytoplankton concentrations. The logged concentrations could then be transmitted on a regular basis if the system had the capability to transmit data or it could be manually downloaded when the float was visited or retrieved.
For both these groups the algae concentrations had to be measured in a way that would be possible on site (in situ) and provide immediate measurements. The measurements had to be
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Chapter 1 Introduction
done without influencing the physiological state of the algae or phytoplankton or disturbing the environment around them. The trend of the algae or phytoplankton concentration over time can then be investigated. This trend provides valuable information, like the overall conditions of the water around the algae or phytoplankton, and can indicate events like pollution since population growth has been shown to be susceptible to toxic pollutants [1].
Algae and phytoplankton both contain chlorophyll that they use to turn light into energy. Chlorophyll fluoresces (emits light) when light is shining upon it. This is discussed in much more detail in Chapter 2. According to literature [2],[3],[4] the fluorescence intensity of chlorophyll is proportional to its concentration in the water. Fluorescence meters (fluorometers) that measure chlorophyll fluorescence and can determine chlorophyll concentration have been built by [5],[6],[7],[8] and [9]. Our hypothesis was that it would be possible to build a fluorometer from low cost components that could be used as a chlorophyll concentration sensor. Measuring the chlorophyll concentration would then enable the calculation of the algae or phytoplankton concentration when the chlorophyll concentration of each species is known. It was not intended to add other functionality to the fluorometer, like measuring quantum yield (number of photons emitted/number of photos absorbed) or performing biomass calculation.
A fluorometer induces fluorescence by shining an excitation light on an area or object. In the case of this project the fluorometer excites fluorescence by shining the light into a fluid containing the algae or phytoplankton. The fluorometer then measures certain fluorescence parameters, like fluorescence intensity, to calculate the chlorophyll concentration that in turn can be used to determine the algae or phytoplankton concentration. Beutler [10] did groundbreaking work on concentration measurements and developed a research fluorometer in 2003 that could determine algae and phytoplankton species composition and concentration from chlorophyll fluorescence.
Schreiber has developed commercial fluorometers for photosynthesis research since 1986 and has published many articles about chlorophyll fluorescence and its measurement [11]–[18]. Currently there are many commercial fluorometers available that make use of expensive and sometimes specialised components that drive up the price of the product. These fluorometers usually also have added features that were not required by the CSIR or CeBER for the intended use as an algae or phytoplankton concentration sensor. The development of these added features also drives up the cost of such fluorometers.
The fluorometer developed during the thesis project is called the FICC (Fluorescence Intensity
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Chapter 1 Introduction
Chlorophyll Concentration). A few requirements were decided upon at the start of the design phase of the FICC. As the project mainly started with the aim of developing a low cost fluorometer, the target was to keep the total cost of the system below $1500. Only low cost commercial components that are freely available would be used in the design. This would provide a fluorometer with a cost that is a tenth of the cheapest commercial product currently being used by the CSIR. Such a low cost would make it possible to deploy several of the fluorometers on the submersible floats for long periods in dams and in the ocean along the coastline without a major financial risk of the fluorometer getting lost or damaged.
To measure the phytoplankton concentrations in the ocean, the fluorometer had to be able to measure phytoplankton concentrations as low as 0.1 µg/l. These submersible floats would run on batteries. Consequently the fluorometer had to be designed with low power consumption as a high priority. The size of the fluorometer also had to be kept as small as possible as there was little space available on the submersible float. The target shape of the fluorometer housing was a cylinder with a diameter of 30 mm and length of 150 mm. The fluorescence measurement section ideally had to have a flat contact surface with the fluid containing the algae or phytoplankton. This would make it easier to clean with a wiping mechanism while in use on the float. The housing had to be waterproof down to a depth of at least 5 m as the submersible float it was going to be fitted to could dive down to this depth.
1.2 The Concept Design
During the development of the FICC, many prototypes of the subsystems were built to investigate the comparative performance of components as well as the response of chlorophyll to various light conditions. Some of these subsystem prototypes are discussed in the thesis chapters that cover the different components and lighting methods that were tested. Even though the requirements of the FICC indicated a final product with a flat face that can be placed in the fluid containing the chlorophyll, almost all the prototypes were built around the concept of having a cuvette holder in a “front end” subsystem where some components were mounted to the cuvette holder. This ensured that there was no change in location of sensors between measurements. A cuvette containing different chlorophyll fluid could then be easily placed and removed in the cuvette holder. This was originally done since it did not require a waterproof housing, which was not available.
This front end that was designed around the cuvette had other advantages during the development period. The front end could be enclosed to keep out all light, other than the excitation light. This
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Chapter 1 Introduction removed the influence of external light, which can distort measurements. Placing the components around the cuvette holder enabled the testing of different design ideas without requiring a redesign of the housing. Making use of a cuvette also allowed the preparation of several different chlorophyll concentrations, consisting of about 5 ml each in different cuvettes, that could quickly be swapped to compare measurements. A flat faced unit would have required larger quantities of chlorophyll fluid to place the FICC in. It would also have required a good cleaning procedure between measurements to prevent cross contamination of samples. This would have taken extra time and introduced the added risk of cross contamination. The diagram below shows the final FICC system design that was used. It also shows the front end components that are located directly around the cuvette holder.
Figure 1: FICC system diagram
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FluorometerAlgaeChlorophyllMeasurementsFluorescence