UPTEC F 16062 Examensarbete 30 hp Februari 2017
Construction, programming and testing of measurement equipment for microbe culturing in space - Contribution to the MOREBAC experiment, part of the MIST-project
Oscar Årling Abstract Construction, programming and testing of measurement equipment for microbe culturing in space
Oscar Årling
Teknisk- naturvetenskaplig fakultet UTH-enheten Many different bacteria have essential roles in the process of recycling organic waste, making them useful tools when it comes to Besöksadress: establishing artificial ecosystems, a key technology to master in Ångströmlaboratoriet Lägerhyddsvägen 1 the expansion of human space travel. Hus 4, Plan 0 In order to further investigate bacteria growth conditions during Postadress: space travel, the MOREBAC experiment was formulated. The objective Box 536 751 21 Uppsala was to design an experimental setup and develop measurement equipment with the capability of confirming successful Telefon: resuscitation of freeze-dried bacteria in space by measuring 018 – 471 30 03 bacteria growth, on-board the student-built MIST-satellite.
Telefax: 018 – 471 30 00 The experimental setup prototype consisted of an acrylic chip wherein the bacteria would be placed during experiments and an Hemsida: optical measurements configuration using a photosensor with the http://www.teknat.uu.se/student purpose of detecting bacteria cell growth. For experimental environment monitoring, a temperature sensor and a pressure sensor were calibrated. An Arduino Nano microcontroller was programmed to control all electrical components during measurements. During the optical density measurements blue dyed water and E.coli bacteria in nutrition media were used as test samples. Provided varying blue dye or bacteria cell concentrations, in the form of dilution series and growth-over-time-series, the equipment proved capable of producing measurements that indicate the optical density of the test sample. Furthermore, a prototype experiment protocol simulating events that will occur in the final experiment design, was implemented and was able to produce real-time monitoring graphs of optical, temperature and pressure measurements, as well as documentation of all events and measurement data.
Handledare: Håkan Jönsson Ämnesgranskare: Maria Tenje Examinator: Tomas Nyberg ISSN: 1401-5757, UPTEC F 16062 Summary in Swedish
Ända sedan den första månlandningen har en av mänsklighetens stora utmaningar och strävanden varit att erövra större delar av universum. Att kolonisera andra himlakroppar är ingen lätt uppgift när dessa inte ens tillåter växter att grönska eller har en atmosfär som tillåter oss att andas. Men det som inte nns kan man alltid försöka skapa. Så, för att vi människor någonsin ska kunna kolonisera andra planeter behöver vi kunna återskapa den omgivning som jorden erbjuder så pass likt att vi faktiskt kan överleva i den. För att skapa en sådan omgivning krävs många olika komponenter för att systemet inte ska fallera. I ett fungerande ekosystem krävs bland annat organismer som kan hantera avfall genom nedbrytning som därigenom tillåter andra organsimer att återanvända avfallet. En mycket viktig komponent i alla ekosystem är bakterier, just på grund av deras delaktighet i nedbrytningen. Bakterier kommer i många olika format och i deras mångfald nner man att de kan uträtta många olika utgifter. Medan vissa bakteriearter orsakar sjukdomar, nns det andra som fullbordar viktiga kretslopp i naturen som exempelvis kväve- cykeln. På KTH pågår projekt kallat MIST (MIniature STudent satellite) där tanken är att konstruera en satellit vari sju olika experiment ska utföras under satellitens omloppsbana kring jorden. Ett av dessa experiment är MOREBAC, som går ut på undersöka odlandet av bakterier i rymden, närmare bestämt frystorkade bakterier och hur väl man kan lyckas återuppliva dessa från deras dvala. I detta examensarbete har målet varit att konstruera mätutrustning för MORE- BAC:s räkning. Mätutrustningen ska tjäna syftet att kunna identi era huruvida koncentrationen bakterier ökar efter återupplivningsförsöket som kommer att ske från frystorkad form. Dessutom ska utrustningen kunna kunna mäta relevanta förhållanden i omgivningen, i detta fall temperatur och tryck. Alla elektriska komponenter, såsom sensorer, resistorer och LED kopplades till en Arduino Nano microcontroller, som programmerades via datorn och sedan kunde
3 man via datorn skicka uppgifter till microcontrollern som i sin tur styrde de elek- triska komponenterna så att de utförde dessa uppgifter. För att kunna odla bakterierna måste vi ha någonting att odla dem i, något förslutet som inte tillåter bakterierna att yga omkring hursomhelst, vilket de gärna gör när gravitationen är liten. Vi använde ett genomskinligt chip med en in- loppskanal där näringsämnen till bakterierna kunde pumpas in och en utloppskanal för att kunna pumpa ut gas och vätska. Eftersom vi vill kunna bestämma bakteriekoncentration utnyttjade vi det faktum att bakterier absorberar mer ljus ju högre koncentrationen är. På så sätt kunde vi använda en ljuskänslig sensor till att bedömma hur stor andel ljus bakterierna absorberade när de belystes med en riktad ljusstråle från en LED. Exempelvis, om en liten mängd ljus skulle släppas igenom bakterierna betyder det att de ab- sorberar en stor andel av ljuset, vilket tyder på en hög koncentration av dem. Mätningar på olika bakteriekoncentrationer gav oss därmed en bra referens till vilka mätvärden som motsvaras av en viss koncentration. Inledningsvis, för att testa den principen och för att undvika tidskrävande bakterieodlingsförberedelser, testades mätutrustningen på olika spädningar av färgat vatten. Temperatursensorn och trycksensorn testades för olika temperaturer respektive trycknivåer och påvisade båda två att de var linjärt beroende av mätvärdena utan märkbara avvikelser, vilket innebar att de kunde anses som tillförlitliga för refer- ensmätningar av dessa storheter på intervallen 25 ◦C till 70 ◦C, respektive -80 kPa till 30 kPa. När alla komponenter hade testats separat testades de alla samtidigt enligt exper- imentprotokollet, vilket innefattar alla händelser som kommer ske när satelliten är satt i bruk och det är dags för experimentet att utföras. Under testutförandet av experimentprotokollet skapades, samtidigt som mätningarna pågick, en visualiser- ing med tre grafer föreställande mätningar på absorbans, temperatur och tryck. Dessutom dokumenterades alla händelser i en l på datorn. Från resultatet av utförandet av experimentprotokollet kunde det konstateras att händelserna dokumenterades som de skulle och att mätutrustningen kunde följa temperatur-, tryck- och absorbansförändringarna som skedde. Dock antydde ab- sorbansmätningarna ett temperaturberoende, vilket innebär att den valda ljussen- sorn omkalibreras eller bytas ut mot en ljussensor utan temperaturberoende. I fortsättningsarbetet kommer det behövas försök till återupplivning av frystorkade bakterier, utredning och åtgärd för ljussensorns temperaturberoende samt paral- lella experimentutföranden, med varierande parametrar så som tidsperiod, djup på chippets bakteriebrunn.
4 Contents
1 Introduction 7 1.1 Aim of Project ...... 7 1.2 Bacteria in Space ...... 7 1.3 Life Support Systems and Pocket Earth Ecosystems ...... 8 1.4 Detecting Bacteria Growth ...... 9 1.5 Studies of Previous Work ...... 11
2 Background 13 2.1 The MIST satellite project ...... 13 2.2 MOREBAC ...... 14 2.3 The Employer ...... 14
3 Theory 15 3.1 Experiment Limitations ...... 15 3.2 Serial Communication ...... 16 3.3 Experimental Preparations and Procedure ...... 16 3.4 Experiment Protocol ...... 17
4 Design, Construction and Testing 20 4.1 Electrical Components ...... 20 4.1.1 Microcontroller and Computer Softwares ...... 22 4.1.2 Optical Measurement Components ...... 23 4.1.3 Temperature and Pressure Sensor Calibration ...... 24 4.1.4 Choosing Resistances ...... 25 4.2 Chip design ...... 26 4.3 Optical Measurement Con guration ...... 28 4.4 Arduino and Processing programming ...... 29 4.5 Dye and Bacteria Measurements ...... 30 4.6 Experiment Protocol Testing ...... 31
5 Results 32
5 5.1 Resistance tests ...... 33 5.1.1 Analytical Resistances ...... 33 5.1.2 Typical Photoresistance Measurements ...... 35 5.2 Dye and Bacteria measurements ...... 36 5.3 Temperature and Pressure Calibration ...... 39 5.4 Experiment Protocol ...... 40 5.4.1 Logged Events ...... 41 5.4.2 Real Time Monitoring Graph ...... 42
6 Discussion 44 6.1 Conclusion ...... 46 6.2 Future work ...... 47 References ...... 48
6 Chapter 1
Introduction
In this section, the underlying circumstances that motivate the MOREBAC-experiment are presented, such as the usefulness of bacteria in life support systems and arti - cial ecosystems. Also, the aim of the project, what approaches were taken to get there, and design limitations are all discussed.
1.1 Aim of Project
The aim of this master thesis was to develop measuring equipment for the detection of growth of resuscitated freeze-dried bacteria, on-board a student built satellite orbiting Earth. Initiating the MOREBAC project, this rst phase of the project served to establish a basis for other students to continue working on to reach a nal product that will be able to be incorporated into the MIST-satellite.
1.2 Bacteria in Space
Bacteria can be useful in many appliances related to the recycling of biological waste, which would be particularly crucial on space missions where the resources that are brought along must last throughout the duration of the mission. Bringing bacteria out into space requires rigorous con nement to avoid any con- tamination on-board the vessel. Furthermore, the bacteria are living cells that require nutrition and the right environment to thrive.
7 One suitable solution to the need of keeping bacteria alive during long space mis- sions, is to use freeze-dried bacteria that are resuscitated when the moment is right for the bacteria to be put into use. However, this requires a reliable resusci- tation method where enough bacteria survive and are able to reproduce. To verify that the bacteria indeed survive the process of resuscitation, measuring equipment capable of detecting the bacteria growth is needed.
1.3 Life Support Systems and Pocket Earth Ecosys- tems
Why do we need humans operating in space? Why can't we just have robots performing the work of humans? Robots are powered by electricity, and unlike us organic humans, need no nutrition intake and do not have as strict environmental requirements. Furthermore, robots have no juristic rights and thus can be used for tasks that would put a human in harm's way. However, there are many reasons why humans should be sent into space, and not just robots. For one, sending humans into space helps improving our understanding of how the space environment aects the human body. Humans are also more apt at solving a larger variety of tasks as we can use our knowledge and problem- solving skills to nd solutions to any problems that occur. So far, no arti cial intelligence has been developed that can replace that ability. The machines are primarily tools to save time, reduce danger, and increase precision. When humans are sent out on space ight missions there is no guarantee that they will survive the trip, but there is however an enormous eort put into lowering the risks of all forms of exposure to danger. There is always a plan to bring the astronauts back to Earth alive after the mission's completion.[1] The rst moon landing proved that humans can actually visit other celestial bodies and return safely. Knowing this is possible, it is not an unthinkable notion taking it to the next step - staying for a longer period of time, and ultimately even colonize other planets. But to do this, we would have to be able to create a pocket Earth ecosystem: An enclosed volume with arti cial atmosphere resembling the Earth's, where humans and other life forms can live without wearing space suits or carrying any life aid. In order to create a ecosystem that sustains an environment in which humans can survive, there are many conditions that must be ful lled. For example, the air composition must be regulated so that the air remains breathable instead of
8 becoming choking or toxic. Other vital requirements are clean water, food with essential nutrients, livable temperature and pressure levels, and protection from radiation.[2] Normally, all this is available beneath Earth's atmosphere but during space missions all that is available are the things that are brought along aboard, therefore a substitute for all the essentials Earth provides has to be presented. The system put in place to enable humans to survive during space ights, by regulating the environment and supplying life-necessities, is called a life support system. Imitating the conditions found in Earth's atmosphere, a life support system of a pocket Earth ecosystem would have to be very immaculately designed. One of the most important aspects of a sustainable pocket Earth ecosystem is the recycling of biomaterial, and this is where bacterias play a substantial role. In the process of biomaterial recycling all waste is decomposed, which can then be reused as nutrition for organic life forms such as plants and bacteria. However, all cycles need to be closed for the system to be able to be sustained without introducing new material to the system. An ecosystem should, after a long period of time, show no signs of having essential building blocks ending up amassed and unable to partake in the circulation.[3] When organisms die, certain bacterias and fungi that exist in the soil decompose the proteins into smaller elements. After several steps in the ecosystem's chain of possible processes, the elements nd their way back to where they once started with the help of bacteria, other living organisms and also things like dierent weather phenomena. In the nitrogen cycle, a number of bacterias interact with nitrogen compounds in dierent ways. Decomposing bacteria breaks down organic waste material into ammonia and ammonium ions, and nitrogen xing bacteria x nitrogen gas from Earth's atmosphere into the soil. Denitrifying bacteria helps release nitrogen back into the air. Together, these bacteria are essential in creating a balance of nitrogen in its varying compositions. [4]
1.4 Detecting Bacteria Growth
The main focus of this thesis was to develop a method of veri cation for bacteria survival and reproducibility after the resuscitation from freeze-dried form. To measure bacteria growth, suitable techniques had to be considered from pre- existing techniques. One technique which is prevalent in laboratories of biological research (by the use of spectrophotometers) is measuring the portion of light that
9 passes through a substance containing the bacteria. The substance's light trans- mission rate is referred to as optical density, a property that relates to the how much light the particular bacteria species absorbs, and its concentration, thus making this technique an excellent approach for detecting bacteria growth. With access to the right nutrition and environment, a bacteria cell divides peri- odically into two. The new cell is identical and will also reproduce in the same fashion. This means that, as long as the growth conditions are right, a population of bacteria cells will continuously duplicate at a certain time interval. E.coli was used as the subject bacteria in this thesis work, preferred for its speedy growth rate. Depending on the environment, the duplication rate of E.coli can vary immensely, from 20 minutes in optimal conditions to a much slower rate of 12-24 hours which is its estimated intestinal tract duplication rate. [5] The life-cycle of bacteria is usually divided into four phases that are characterized by the bacteria cell concentration curve slopes, from the culturing of a batch performed in a closed system. Initially, during the lag phase, the bacteria prepare to reproduce and have not yet started multiplying noticeably. Then, during the exponential phase the bacteria growth skyrocket until the resources starts to thin out. Next, as the growth is suppressed in the stationary phase, the bacteria stop growing in numbers and stay at a steady level. Finally, in the death phase, the cell population declines as a result of cell-deaths happening more often than cell-division, due to all the toxic metabolic waste accumulated during the span of the bacteria cell culturing, as well as the shortage of nutrition and oxygen. [6] Even though the number of living bacteria cells decrease during the death phase, absorbance measurements of the culture will not yield lower value results as even dead cells contribute to a higher absorbance, meaning that the absorbance mea- surement will indicate the concentration of the sum of living and dead bacteria cells.
10 1.5 Studies of Previous Work
In preparation of the thesis work, similar studies that have been performed involv- ing monitoring of micro-organism growth were studied. In the paper Growth monitoring of a photosynthetic micro-organism (Spirulina platensis) by pressure measurement, estimations of concentration levels of the pho- tosynthetic cyanobacteria Spirulina plantensis were made through pressure mea- surements. Much like in the MOREBAC experiment, the bacteria were cultured inside a closed system. Since the photosynthesis of the cyanobacteria resulted in continual release of oxygen, the pressure levels were constantly increasing, requiring pressure regu- lation every now and then, when a certain pressure level was reached. Therefore, pressures changes had to be tracked cumulatively to account for the release of pressure due to regulation. The method of using pressure measurements to monitor the concentrations was validated using physiological models that involved the cell metabolism and its material balance relations during growth and its eects on the pressure inside the closed volume.[7] This technique could be a good compliment to the absorbance measurements in the MOREBAC experiment, since pressure measurements will be performed re- gardless, as it will be necessary for pressure regulation. However, determining the correlation between concentration and pressure will require models that describe all the physiological circumstances of the growth process well, which will depend on what bacteria that will ultimately be the subject for the experiment. For example, factors like pH might be necessary to know in order to calculate the concentration, as was the case for the Spirulina plantensis culturing measurements. This would require more measuring equipment, testing and research, which could be di cult to t in the frame of the MOREBAC experiment. Another study, Design, Operation, and Modeling of a Membrane Photobioreactor to Study the Growth of the Cyanobacterium Arthrospira platensis in Space Condi- tions, compared dierent methods of measuring micro-organism growth in space conditions. The methods tested were optical density, pressure and pH measure- ments. The experiment was carried out over 500 hours to what long-term eects radiation had on measurements. Like in the previously discussed study, pressure measurements were conducted to deduct bacterial growth. To make sure that the pressure sensor was measuring on oxygen, a hydrophobic membrane was used to separate the oxygen from the
11 liquid phase. Pressure increased as a consequence of oxygen evolution during the photosynthetic process, during which the pH increased as well due to carbon consumption, providing yet another way to indirectly measure bacterial growth. However, to maintain cultivation, the pH-value had to be regulated when the levels started to inhibit growth. Keeping the pH in the range of 8.3-10, carbon dioxide
(CO2) was supplied to the culture to bring the pH-level back down. In the context of the MOREBAC experiment, the pH-measurements would be di cult to perform since additional operations involved in regulating pH, as well as putting a pH-probe in contact with the bacteria growth chamber would be complicated, requiring additional channels in the chip. When it comes to the long term eects of radiation on the equipment, the compo- nent used for optical density measurements, a photodiode, could only give reliable measurement data up to 300 hours into the experiment. In other words, the optical measurements became inaccurate over time, possibly due to radiation exposure. However, pressure measurements did keep producing reliable measurements data throughout the whole duration of the experiment, with accurate and precise re- sults. [8]
12 Chapter 2
Background
This section familiarizes the reader with the context in which the work has been carried out.
2.1 The MIST satellite project
All over the world, researchers and university students design and build small satellites, called CubeSats, which are sent into orbit around earth once they are operational and certi ed by the space agency of the a liated region. The MIST- satellite (Mini Student satellite) project is a cooperative eort among university students to construct a satellite containing several scienti c experiments, which are to be executed inside the satellite once it is orbiting earth. One of these is MOREBAC, which will be the only experiment carrying organic material. The projects have been suggested by KTH Royal Institute of Technology, IRF the Swedish Institute of Space Physics and the two companies NanoSpace AB and Piezomotor AB. Sven Grahn is the project leader. The work started the 28th of January 2015 and is estimated to be nished in 2017, with a launch in 2018. [9] There are seven separate projects which are all developed by student groups. How- ever, since all the projects will share the restrictions of space and power consump- tion, it is imperative to establish a good communication and coordination between the projects for everything to be able to run smoothly after launch.
13 2.2 MOREBAC
MOREBAC (Micro uidic Orbital Resuscitation of Bacteria) was proposed by the Division of Proteomics and Nanobiotechnology, KTH. The MOREBAC experiment will be carried out aboard the MIST-satellite after students have presented their contributions to its development and all equipment is indeed operational. The purpose of the experiment is to investigate the resuscitation process of freeze- dried bacteria in the satellite during its orbit around Earth. MOREBAC will be carried out by master students from KI, KTH, SU and UU, working in succession to develop the experimental setup.
2.3 The Employer
This thesis was carried out at the Department of Proteomics and Nanobiotechnol- ogy at Science for Life Laboratory, SciLifeLab, under the supervision of researcher Håkan Jönsson, who is also a lecturer at KTH Royal Institute of Technology. SciLifeLab, is a center of national resource for molecular biosciences with focus on health and environmental research. SciLifeLab is a collaboration between four universities: Karolinska Institutet, KTH Royal Institute of Technology, Stockholm University and Uppsala University.
14 Chapter 3
Theory
This section further explains the scope of the project, aspects such as limitations, communication, and the experiment formulation.
3.1 Experiment Limitations
The space environment and the size of the satellite bring about limitations for all the MIST-satellite projects. Each project has been allotted a partition of the total space inside the satellite. The alloted space for the MOREBAC experiment is ap- proximately 70x70x40 mm, but these dimensions are subject to change depending on whether other experiments will need more space inside the satellite.[10] Furthermore, because of the limitations on the power usage, the equipment needs to have low voltage levels and low power consumption to be able to hold up to the shared capacity. A lower power consumption also means better temperature control since less heat will omit from the equipment during use. The maximum voltage the components will be supplied with will be limited to 5 V. Another restriction that will have to be taken into account is the materials. For example, some plastics are prone to out-gassing, which is a slow evaporation of the material, emitting gases to the nearby environment. This is mainly a problem when the material is exposed to vacuum.[11] Materials that are porous should be avoided when it comes to making the culturing chip, since all gases, uids and of course the bacteria themselves need to be contained and not leaked out. Furthermore, unless a covering layer that protects the equipment from radiation exposure is implemented, the components and materials must be able to withstand radiation.
15 3.2 Serial Communication
Microcontrollers are helpful tools that enable communication between computer software and electrical circuitry. Commands can be sent from the computer through a serial cable informing the microcontroller which of the connected com- ponents should be active and for how long. The information ow goes in both directions also allowing the computer to access measurement data from the mi- crocontroller. The work in this thesis heavily depends on the microcontroller's central role in the testing of electrical components and the monitoring of the bac- teria growth experiments.
3.3 Experimental Preparations and Procedure
For some of the measurement equipment testing, a sample substance containing bacteria was required and therefore the sample had to be prepared before any measurement could be initiated. The culturing of E.coli bacteria was conducted in a biology lab where many dier- ent bacteria were handled on a daily basis. Consequently, the risk of contamination had to be taken into account when handling the bacteria samples, because if other bacteria started growing in the sample, the measured absorbance would not be that of the subject bacteria. So, when a sample container was exposed to the open air, a burner was used around its edges to mitigate the risk of contamination by killing o other bacteria that might have lingered in the air. To start the culturing process, the original batch of E.coli were taken out from the freezer, and from the batch a small amount of bacteria cells were extracted using a cell spreader, which is a long thin plastic tool that facilitates bacteria extraction. The cell spreader with the attached bacteria was used for stirring in a plastic container containing nutrient media so that the bacteria would mix into the media. Then the container was put into an incubator so that the bacteria could thrive and multiply. The incubator was constantly shaking the bacteria sample with a rotating motion, while keeping the temperature at a constant 37◦C. After 8 hours in the incubator, the E.coli had grown so much that they visibly clouded the media. Thus, the media with these bacteria had become a quali ed candidate for the base sample for dilution and growth experiments. Once the base concentration sample had been produced, it was time for either a dilution series or a time series to be measured in the optical measurement device
16 prototype. For absorbance calibration of the dilution series, each dilution's op- tical density was also measured using a spectrophotometer. For the time series, the optical density was measured in the spectrophotometer just before starting and after nishing the constructed measuring device measurements. The spec- trophotometer measurements were performed by putting the samples in cuvettes, small measuring jugs with square-shaped cross sections, which were put into the spectrophotometer. Using a syringe and tubes, the sample that was going to be measured was injected into the chip through the inlet channel and the chip was placed in measurement position, between the mechanical iris and the photosensor, with its surface perpen- dicular and centered to the light beam that would be emitted by the LED during a measurement. To be able to produce any measurements, the mini-USB had to be connected to the Arduino Nano and the computer and then the Arduino script uploaded onto the microcontroller. Lastly, the measurement device was placed into a light-impenetrable carton box and the Processing script was started. The monitoring process had begun and measurements were being stored on le.
3.4 Experiment Protocol
Experiments that will be performed during the satellite's orbit must be thoroughly tested on earth beforehand since there is only once chance for each experiment to be executed after the satellite's launch. Therefore a protocol for the experimental procedure needs to be designed and tested. Every event that takes place in prepa- ration of and during the experiment should be documented over the timespan of the experiment along with the measurement data. Also, before and during the experiments, a number of variable checks and actions such as temperature and pressure control must be performed to ensure that the right experimental conditions are ful lled. All these events need to be part of the protocol. At the nal stage of the project, when the measuring equipment development is nished it will be mounted onto the MIST-Satellite and be connected to the main computer from where all commands of the protocol will be sent, and to where all measurement data will be sent back. During the bacteria growth detection experiment, many dierent events occur.
17 Some are periodically reoccurring and some only happen once. For example, mea- surements of absorbance, temperature and pressure need to be performed through- out the whole experiment while events like injection of media into the bacteria chip only takes place once. The experiment protocol script handles all these events while storing important information from the whole process, so by implementing it the experiment can be realized and more easily analyzed. Here is a preliminary version of how the experiment protocol looks. Note however that the only events that could be carried out were the actual measurements, and no mechanical or thermal events such as temperature and pressure regulation or in ow of medium. The events that take place during the experiment are described below.
Temperature control: Monitor the temperature and regulate it, to ensure the right conditions for the medium to be uid before injection into chip and for bacteria to grow. - Temperature measurement at a close proximity of the chip's culturing chamber. - Temperature regulation, if temperatures are out of acceptable range. In ow: Injection of medium, containing nutritions and resuscitation liquid, through the chip inlet channel into the culturing chamber. - Open chip inlet and outlet valves - Injection of medium - Close chip inlet and outlet valves Pressure control: Maintain a pressure level that is suitable for the bacteria to grow in and that does not strain the chip, as the pressure increases when gases are released in the cell division process. Repeat following steps until pressure level is on that level. - Measure pressure level at chip outlet - If the pressure level is too high, open chip outlet valve for a short duration and then close it
18 Absorbance measurement: Monitor bacteria growth through absorbance measurements - Measure optical density
Here follows the order in which events will take place during the experiment: 1. Temperature control, performed periodically throughout the experiment 2. In ow, performed once to initiate resuscitation process 3. Pressure control, performed periodically throughout the experiment 4. Absorbance measurements, performed periodically throughout the ex- periment Note that after the in ow of medium, the pressure control and absorbance measure- ments are initiated and continue periodically with independent time steps.
19 Chapter 4
Design, Construction and Testing
In this section, the development and testing process is presented. This concerns the choices of electrical components, the design of the bacteria chip, the con guration of the optical measurement device, the programming of microcontroller as well as the execution of measurement tests.
4.1 Electrical Components
From a large online selection of electrical components, the appropriate sensors, LED:s and resistors were selected and ordered. All selected components adhered to the 5 V restriction of the experiment and can be seen in Figure 4.1.
20 Figure 4.1: Electrical components used to construct measuring device. a: Micro- controller Arduino Nano (Digikey, A000005), b: LED (Thorlabs, LED591E), c: Resistors (1.2 kΩ, 10 kΩ), d: Photosensor (Digikey, Parallax Inc. 350- 00009), e: Temperature sensor, (Digikey, MCP9700A-E/TO), f: Pressure sensor (Digikey, MPXV5100DP)
The testing of the electrical components of the measuring equipment was done on a wiring breadboard, a board with a grid of pinholes for connecting loose end wires and pin components. How all the components were connected can be seen in Figure 4.2.
21 Figure 4.2: Schematic of all electrical components used
In the measuring device prototype, the microcontroller was connected with the electrical components in such a way that four dierent outputs from the micro- controller could activate either the LED, the photosensor, the temperature sensor or the pressure sensor. Also, connections were made from the photosensor, the temperature sensor and the pressure sensor to the microcontrollers input pins. These connections established measure points for the sensor measurements.
The LED was serially connected with a 1.2 kΩ resistor, which is called voltage division, preventing the voltage from overloading the LED. Similarly, the pho- tosensor was serially connected with a 10 kΩ resistor. This voltage division was necessary for creating a new measuring point, since a voltage drop depending on the current of the circuit would occur over the serial resistance. Otherwise, with- out the resistance, we would just be measuring the output of the microcontroller when measuring over the photosensor. Thus, with the new measuring point, the voltage over the photosensor varied whenever its resistance or current varied. The con guration can be seen in the schematic in Figure 4.2.
4.1.1 Microcontroller and Computer Softwares
An Arduino Nano, see Figure 4.1 .a, was used as microcontroller to control the other electrical components of the measurement device. It was mounted onto the breadboard and connected with the components as can be seen in Figure 4.2.
22 The microcontroller has 32 pins, is capable of supplying an output voltage of 5 V and its input resolution is 5/1024 V. Additionally, it has mini-USB socket for computer connection. the Arduino software was used to program the behavior of the microcontroller. In the software, output voltage of the microcontroller's speci ed pins can be activated, as well as input data from the pins connected to a circuits measure-points. Processing, is a java-based software that provides an easy-accessible graphics user interface that by default uses a drawing loop for visualization. It can also be used to send and receive serial information and thereby communicate with the microcontroller. It was used during measurements, sending commands to activate electrical components at speci c times as well as storing and visualizing measure- ment data.
4.1.2 Optical Measurement Components
For the optical density measurements, apart from the serial resistors in the voltage divisions, two electrical components were required: An LED, as seen in Figure 4.1.b, to emit light, and a photosensor, as seen in Figure 4.1.d, to measure incoming light. Additionally, the wavelength of the light emitted from the LED needed to be able to be detected by the photosensor as well as be absorbed by the subject bacteria depending on its concentration. A commonly used wavelength for E.coli absorbance measurements (and many other bacteria) is 600 nm, an orange-yellow color. Wavelengths within the visible light spectrum are also less harmful for the E.coli than for example UV-rays.[12]. Among the LED:s with a range of wavelengths close to 600 nm, an LED of 591 nm was selected for the optical density measurement testing. The spectrum of the LED tted into the intended bacteria's light absorption spectrum as well as the photosensor's detection range. To be able to perceive a small change in light level, which would imply bacteria growth in an absorbance measurement, the photosensor had to be sensitive at the interval of change. Furthermore, since the microcontroller only has a resolution of 5/1024 V, the range of the measured voltages needed to be as large as possible from the lightest to the darkest light levels of the measurements in the experiment. Based on these criteria, the photoresistor seen in 4.1.d was chosen to be used as photosensor for the measuring device. The photoresistor is characterized by having a resistance that is light-dependent, going from a very high resistance at low light
23 levels to a much lower resistance at high light levels. That resistance change corresponds to a voltage change over the component, which can be measured and used as an indicator for absorbance.
4.1.3 Temperature and Pressure Sensor Calibration
Some bacteria are known to not only withstand high temperatures, but even pre- ferring them. Extremophiles, as they are called, have optimal life conditions in temperatures where humans could not live. For example, Thermus Thermophilus which has a 68 ◦C optimum.[13]. E.coli however, along with the majority of bacteria species, prefer milder temper- atures. E.coli thrives in the human intestines where its optimum temperature of 37 ◦C is found. The maximum temperature at which E.coli keep growing depends on what bacteria strain the particular belongs to. Generally the growth is inhib- ited at 41 ◦C. [14] E.coli can survive freezing temperatures, and can even grow at low temperatures, although at a very slow rate, like 7 ◦C. [15] However, in the MOREBAC experiment, the aim is to maintain the temperature around its optimum growth rate temperature. To maintain temperatures and pressure levels within the right intervals, the sensors with the purpose of measuring those quantities had to be calibrated, in addition to the regulators acting to keep the levels in check. However, no testing of regulators was performed at this early stage of the MOREBAC project.
The calibration of the temperature sensor which stated range is −40◦C ∼ 125◦ C was performed by putting the sensor in a block heater (SBH130, accuracy ± 1 ◦C) that provided temperatures from 25◦C to 70 ◦C. [16] All voltages measured by the temperature sensor could be plotted to the corresponding temperature, thus using calibrated values from these measurements can later produce temperature values from measured voltages in the environment of choice, such as in the MIST CubeSat. The pressure sensor has two ports onto which tubes can be connected and a voltage is measured that relates to the pressure dierence. Similar to the temperature calibration, the pressure calibration was performed by having a pressure giver (PC20•25) providing pressure levels ranging from -80 kPa to 30 kPa, measured with a ± 2.5 % accuracy.[17] This range allows the pressure to be measured for uctuations in one direction up to 80 % of the atmospheric pressure, 101 kPa, which will be the operating pressure inside the chip during the experiment.
24 4.1.4 Choosing Resistances
Besides the innate resolution limitation of the microcontroller, by the 1024 mea- suring points available, there are ve major factors that aect the resolution of an optical measurement. These factors are the photosensor, its serial resistance, the LED, the chip thickness and the absorbance of the test sample at its maximum. Considering that the maximum absorbance of the test sample will likely have a typical value at the bacteria cell death phase, and that the chip characteristics cannot be varied easily without having to mass produce all the alternative chip models, a reasonable way of ne-tuning the resolution was to decide on an LED and photosensor and then test for varying resistances. Depending on the maximum light exposure of the photosensor, the resistance con- nected in serial had to be chosen with consideration to that. In the experiment, as it would be performed aboard the satellite, the typical light level at maximum exposure would correspond to the case where the chip and freeze-dried bacteria are placed between the LED and the photosensor at the stage of the experiment where the resuscitation process has not yet begun. However, instead of calculating the typical maximum light level, the typical resis- tance of the photoresistor at the maximum light exposure could be used since the resistance of the photoresistor changes with exposure to light. Having a voltage division over the two components in serial, the resistance of the photosensor, Rp, could be calculated by:
R = R p Vs −1 Vp where R is the serial resistance, Vs the supply voltage and Vp the voltage over the photosensor.
By varying R and measuring Vp, and seeing that the photoresistor's resistance indeed did not change remarkably the measurements indicated a typical resistance around 10 kΩ. After having calculated the resistance of the photosensor at maximum light expo- sure from the LED, the serial resistor could then also be determined. To get a better understanding of how the choice of the serial resistance aected the outcome of the measurements, simulations were made over the resistance span of the photosensor. Also, taking into account what serial resistance would present the highest sensitivity to a change of light level, the derivative of the voltage over the photosensor with respect to the photoresistance was derived. It was found that the derivative was the largest at R = Rp, which meant the sensitivity was the highest for that choice of serial resistance.
25 Formula for the sensitivity of the voltage with respect to a change in photoresis- tance:
dVp Rp = Vtot 2 dRp (R+Rp) A graph of the derivative was plotted for dierent choices of R showing at what resistances the optical measurement device would be most sensitive to a change in light, see 5.1, page 33.
The serial resistance 10 kΩ was decided upon because it was close to the typical measured photoresistance and required no more than one resistor from the resistors available.
4.2 Chip design
A prototype chip in which the bacteria can be placed and start growing after the resuscitation needed to be designed. In order to keep bacteria in place a culturing chamber was designed to be a cylindrical cavity inside the chip, with the axis perpendicular to the chip's top and bottom surface. Additionally, the chamber had to have an inlet for in ow of resuscitation medium and an outlet for pressure balancing when media is injected through the inlet channel and for the bacteria growth phase, which will otherwise result in elevated pressure levels, risking damage on the chip. The inlet and outlet channels were made very narrow, 0.2 mm, to increase the in uence of the shear stress exerted upon ingoing and outgoing uids and thus improving the control of the ow. These channels are connected with the chamber, continue outwards in a direction parallel with the long side of the chip and halfway to the edge they make sharp right-angled turns towards the surface of the chip with the largest area, corresponding to the bottom of Figure 4.3, until they reach the very surface. For optical density measurements it was important for the surfaces of the chip, especially at the bacteria chamber, to be as plane and smooth as possible in order to disperse a minimal amount of the light that is going through the chip. The rst chip prototype was produced at KTH Machine Design prototyping center. The model used for this was created in the CAD-program AutoCAD, where a 3D- model was constructed. From the model, 2D-images were extracted from dierent angles to be used as reference for creating the chip prototype. The chip was divided into layers since the machine drill only permitted drilling from a surface and
26 inwards with a drilling area that could not become larger as the depth increased. Therefore, creating a hole inside a solid block for example would not be possible with the drilling technique available. Had 3D-printing been an option however, which it wasn't because of the budget, there would not have been a need for as many layers, but the fact remains that at least two layers are required to enable bacteria to be placed properly into the bacteria chamber. Screw-holes were required to go through each layer of the chip so that the layers could be assembled by being screwed tightly together. Transparency of the material is absolutely necessary for the optical density mea- surements. Furthermore the chip needs to withstand varying temperatures and pressures. Acrylic was chosen for the rst prototype since it was a transparent and hard material that could be shaped according to the 3D-model design, and was available at KTH Machine Design prototyping center. In future work the chip should be tested for the range of temperatures that it will be exposed to during the experiment aboard the satellite, but to start with testing and further developing of the chip design was in focus.
Figure 4.3: A 3D-model of the rst chip prototype, with screw-holes in each corner, a chamber in the center and an inlet channel as well as an outlet channel going from the chamber.
27 Table 4.1: Chip Dimensions Chip length 76.2 mm Chip width 25.4 mm Chip thickness 2 + 3 + 2 mm Chamber diameter 10 mm crew-hole diameter 5 mm Inlet diameter 0.5 mm Channel width 0.2 mm
In table 4.1, the dimensions of the chip are speci ed. The Chip thickness was later divided into three layers instead of two in order to create a smoother surface at the chamber area. The top and bottom layer were each 2 mm and the middle layer 3 mm.
4.3 Optical Measurement Con guration
It was important to create consistency in the optical measurements in order to attain an acceptable accuracy. The main problems were to focus the LED so that it was directed straight towards the photosensor, to keep the bacteria chip in a locked position where the bacteria would be centered, and also, to have the same ambient level of light at the photosensor for each measurement. In order to establish a steadfast con guration where the LED and the photosensor could be locked in place aligned with each other, parts made for optical experiments were used. Included in the optical setup were two steel beams used for alignment, three block holders enabling mounting of the LED and the photosensor as well as improving the balance, and one mechanical iris for creating a small hole through which the light could pass through and be focused on the bacteria chip chamber. See Figure 4.4. Furthermore, during measurements, the whole con guration was enclosed by a carton box to ensure an ambiance as dark as possible.
28 Figure 4.4: Optical measurement con guration. The black parts, from left to right: Block holder with the mounted LED, another block holder, the mechanical iris, and then a block holder with the mounted photosensor. The chip was placed in a slit made in a piece of cellular plastic that enabled the chip chamber to stand in a steady position in the center of the light beam.
4.4 Arduino and Processing programming
The Arduino Nano microcontroller was programmed in the Arduino software. An Arduino script was written that included the responses of the microcontroller to serial information that would be sent from the Processing script on the com- puter. In the Arduino script, all the output pins of the microcontroller were assigned numbers corresponding to what component was connected to them. These pins were used for supply voltages during measurements, keeping components active or inactive at the right times. Similarly, the microcontroller's input pins were assigned numbers corresponding to what measuring point in the circuit that was connected. Through these input pins the microcontroller collected measurement data which was sent back to be stored on the computer by the Processing script. Before the experiment protocol was implemented, every measurement that needed
29 to be made required the Processing script to be run once. In such a measurement, Processing would send a serial message with information about what action to be performed, the microcontroller would receive the message and perform the speci ed action and send the data back to Processing which would save the data to a le on the computer.
4.5 Dye and Bacteria Measurements
Since bacteria must be cultured before use, and also need delicate handling, using blue dye as a substitute sped up the initial testing phase. But once it had been con rmed that the measuring device could detect a dierence in light level, bacteria had to be tested as well. In the case of the dye experiments, a base concentration of 6 drops of blue color mixed into 100 ml water was diluted down until there was just a hinge of blue visi- ble, each time decreasing the concentration down to a half of the previous sample. For the dierent concentrations, the optical densities were measured in a spec- trophotometer to be compared with the prototype measurement equipment. Bacteria dilutions were performed in the exact same way, except that the base sample consisted of E.coli bacteria that had been cultured overnight in nutrient media. Furthermore, the dilution of the bacteria sample were carried out until the spectrophotometer showed a OD-value (optical density) below 0.1, providing a dilution series ranging from OD = 0 to the optical density of the base sam- ple. The spectrophotometer was calibrated to a sample containing only media, corresponding to OD = 0. In order to determine whether the equipment could indeed detect the increase of bacteria after some time in the incubator, a time series experiment was carried out. This means that absorbance measurements were made periodically on the sample during its growing phase in the incubator. A sample was cultured overnight in an incubator and before injecting the sample into the bacteria chip it was diluted with more medium down to a tenth of the previous bacteria concentration, to make sure that it was supplied with enough nutrients and started on a low concentration.
30 4.6 Experiment Protocol Testing
An experiment protocol script was written in both the Arduino and Processing soft- ware, as the Arduino Nano needed to be programmed for the speci c commands that it would receive from the computer. A bacteria growth experiment was per- formed and monitored according to the experiment protocol. In other words, all events were logged and a graph was produced, showing all measurements data. The box containing the measuring device was placed into the incubator and measure- ments were started immediately without letting the box air reach the equilibrium temperature. This was done to be able to see how the temperature sensor behaved as the temperatures slowly increased to the that of the incubator. Also, it enabled us to see whether the other measurements were aected by the temperature level. After about 3.5 hours the box was taken out into room temperature for an hour before stopping the measurements, providing measurements from all the sensors during a decrease in temperature.
31 Chapter 5
Results
In this section, results from the developing process are presented as well as results of measurements performed after the design choices had been implemented. When the chip had been manufactured, all components had been assembled, the microcontroller connected to the optical measurement device and the environ- ment control sensors, the prototype measuring equipment was ready to perform measurements. In order to identify the parts of the measuring device see Figure 5.1.
32 Figure 5.1: Assembled measuring device prototype. A: Pressure sensor with con- necting tube. B: Temperature sensor. C: Microcontroller. D: Bacteria culturing chip. E: Optical measurement device.
5.1 Resistance tests
Both analytical simulations and experimental measurements produced information on what behavior could be expected from dierent choices of resistances.
5.1.1 Analytical Resistances
The graphs made from the analytical formulas show how the choice of serial resis- tance can aect the outcome of the optical measurements, for the whole resistance span of the photoresistor. Over this resistance span and with a supply voltage of 5 V, the graph in Figure 5.2 shows what measured voltages can be expected when choosing one of the three represented serial resistances of varying magnitudes, and the graph in Figure 5.3 show how the sensitivity to a change of photoresistance aects the measured voltages for the candidate serial resistances.
33 Figure 5.2: Simulated results for three Figure 5.3: Change in photoresistance resistances of varying order of mag- for two candidate values of serial re- nitudes for the resistance span of the sistance, R1, for the resistance span of photoresistor. the photoresistor.
In Figure 5.2, it can be seen that for a large serial resistance a substantial pho- toresistance change is necessary to cover the voltage measurement span, meaning small uctuations in light level are harder to detect. Conversely, for smaller serial resistances choices, a large voltage span is covered by a small change in photore- sistance at the highest light levels. For that photoresistance interval a uctuation of light is more easily detectable. Hence, in comparison R = 10 kΩ oers a better resolution than R = 200 kΩ, but is not as linear. However, a linear relation is not necessary since photosensor calibrations will be able to provide a good mapping from measured voltages to optical density, when all components of the experiment have been more precisely de ned. In Figure 5.3, it shows for both the serial resistances that the highest sensitivity to a change in photoresistance can be found where the photoresistance and the serial resistance are the same, since the voltage derivative peaks at those points. This con rms that the resistance can be chosen to give a high sensitivity to a smaller interval by choosing one that is of the same order of magnitude as the typical photoresistance.
34 5.1.2 Typical Photoresistance Measurements
Produced from measurements on the photoresistor for a wide range of serial re- sistances, the following results show the typical resistance of the photoresistor at maximum light exposure during the optical density measurement.
Figure 5.4: Typical resistance of the photoresistor at maximum light exposure, from testing resistances in the interval 10 - 500 kΩ.
As shown in Figure 5.4 the typical photoresistance at maximum light exposure can be expected to be found inside the interval 9.8 kΩ and 11.2 kΩ. In order to minimize the number of resistance components and still remain within this interval, it was decided that a serial resistance of 10 kΩ would be used.
35 5.2 Dye and Bacteria measurements
The dye and bacteria measurements graphs show how well the equipment, after the choice of resistances, can detect a dierence in optical density. The results from the dilution and time series measurements show that the equipment does in fact manage to produce measurements that imply a clear relation between the measured value and the optical densities of the test samples, on the whole span of opacities. In Figure 5.5, the graph shows that the optical density measure- ment equipment can distinguish a dierence in absorbance when subject to blue dye dilutions. The same applies to the bacteria dilution measurements that were performed, showing a similar curve, as can be seen in Figure 5.6. In both these cases, the sample substances were contained in 10 mm thick cuvettes, small-sized measuring jugs with square-shaped cross sections. Even if the optical path length in these measurements was longer than in the nal prototype, the results would at an early stage reveal whether the optical sensor could in fact detect the varying concentrations of the samples.
Figure 5.5: Blue dye dilution measure- Figure 5.6: Bacteria dilution measure- ments with serial resistance of 10 kΩ. ments with serial resistance of 10 kΩ.
Spectrophotometer measurements on E.coli inside the thin chip gave an indication of how well measured voltages correlated to the optical density. This also gave access to optical density calibration data for the thin chip's optical path length in room temperature. Table 5.1 shows the results from the measuring device and spectrophotometer measurements of the bacteria dilution test samples. These results can also be seen in the graph in Figure 5.7, showing how the measured voltages are correlated with the optical densities.
36 Table 5.1: E.coli Dilution Measurements Inside Thin Chip Dilution Rate 1 1/2 1/4 1/8 1/16 1/32 0 Spectrophotometer OD 2.8 1.33 0.75 0.4 0.18 0.1 0 Measured Voltages 2.61 V 2.42 V 2.32 V 2.26 V 2.24 V 2.22 V 2.21 V
Figure 5.7: E.coli dilution measurements: Comparison of optical density mea- sured in a spectrophotometer and voltage measurements made in the prototype measuring device.
Since the bacteria growth measurements were performed with the slightly dierent setup, using a glass chip of 2 mm thickness instead of using the 10 mm thick cuvette, the voltage span became much smaller because of the smaller optical path length decreasing the sample's in uence on the light. This can also be seen in the bacteria growth time series measurement in Figure 5.8, where the curve shows a steady increase in voltage over time, which, in accordance with the dilution measurements, translates to a bacteria cell concentration increase over time.
37 Figure 5.8: Measurements on E.coli growth inside thin chip.
Despite showing signs of growth, the growth rate was expected to be higher because of the E.coli's duplication rate of around 20 min. However, since that rate applies to optimal conditions, and considering that the media temperature was room- temperated at the start of the growth process, the growth rate could not have been as high as at the optimal temperature. Furthermore, since the oxygen supply was scarce, the bacterial growth was inhibited further.
38 5.3 Temperature and Pressure Calibration
Since both the temperature and pressure sensors were tested for two dierent supply voltages, 3 V and 5 V, the results could be compared to see if the lower supply voltage could be a viable alternative in case the power consumption needs to be cut down.
Figure 5.9: Voltage measurements Figure 5.10: Voltage measurements by temperature sensor for temper- by temperature sensor for tempera- atures 25 ◦C to 70 ◦C at supply tures 25 ◦C to 70 ◦C at supply volt- voltage 5 V. age 3 V.
A side to side comparison of voltages measured by the temperature sensor for varying temperatures can be seen in Figure 5.9 and 5.10. The measurements show a linear trend over the interval, making the data reliable to use as reference in other temperature measurements on the same interval.
39 Figure 5.11: Voltage measurements Figure 5.12: Voltage measurements by pressure sensor for pressure dif- by pressure sensor for pressure dif- ferences -80 kPa to 30 kPa at supply ferences -80 kPa to 30 kPa at supply voltage 5 V. voltage 3 V.
A side to side comparison of voltages measured by the pressure sensor for varying pressures can be seen in Figure 5.11 and 5.12. The measurements show a linear trend for the negative pressure dierences, making that data reliable to use as reference in other pressure dierence measurements for that interval.
5.4 Experiment Protocol
During the bacteria growth phase, every occurring event is logged according to the experiment protocol that is executed. Additionally, the monitoring graph generator that was made is updated every time a measurement occurs, showing all temperature, pressure and optical density measurements made since the starting point of the experiment. An optimal time interval between measurements could not be determined since the timings will be formulated from the temperature and pressure regulation system. Readings that are a few minutes apart are enough to enable monitoring of the pro- cess in an environment where temperature and pressure do not vary dramatically and do not need to be regulated often.
40 5.4.1 Logged Events
The experiment protocol script was able to generate a le of logged events with ac- companying timestamps and, in the cases of measurement, measured values. In the example of an experiment run, Figure 5.13, temperature and pressure checks always yields the result that the levels are ne and need no regulation. This is because no regulation of temperature or pressure had been implemented at this stage. Nevertheless, these messages serve the purpose of showing how a typical experiment log le could look like.
Figure 5.13: Real time monitoring log of events from temperature, pressure and optical density measurements during bacteria growth experiment.
41 5.4.2 Real Time Monitoring Graph
The experiment protocol script includes a visualization tool that plots all mea- surements made in the experiment, Figure 5.14. The optical density measurement (represented as absorbance) is displayed in the lower graph in a green color scheme, the temperature as red in the middle graph, and lastly, the pressure as blue in the top graph. As can be seen in all of the graphs, a sudden change of behavior occurs around 210 minutes in. This was caused by the change of environment due to moving the measuring equipment box out from the incubator and into the room. However, by looking at this result an unexpected discovery was made: The ab- sorbance measurement values decreased as the temperature decreased, indicating a correlation between the light sensor's output and temperature levels. This temper- ature dependence could depend on either the LED, the resistors or the photosensor, or all of these components. The temperature interval was realistic as it started at room temperature and rose to the incubator temperature. For each quantity measured, a list of the current, average, lowest and highest values were listed in the information box to the left. In the graphs, it can be seen that both temperature and optical density measure- ments show an increase over time until the change of environment occurred. The slow temperature rise was due to the fact that the box with measuring equipment had to be closed for the sake of not letting any outside light aect the light mea- surements, leading to a slow heat exchange between room temperated air inside the box and the heated air in the incubator. However, the colder room-temperated air from the outside seeping into the box was enough to create a more turbulent air ow inside the box, making pressure uctuations that the pressure sensor measured.
42 Figure 5.14: Real time monitoring graph of temperature, pressure and optical density measurements during bacteria growth experiment.
43 Chapter 6
Discussion
Regarding the data of temperature and pressure calibration measurements, the re- sults show a consistent behavior, making those sensors reliable for measurements in upcoming experiments, where the calibration data trend can be used for reference levels. Even though the temperature calibration measurements deviated somewhat from the mean value of the linear trend that is otherwise present, the results are su - ciently consistent to be used as calibration data for test experiments. At least, until a temperature regulation system that is dependent on the measured temperatures will be implemented. The temperature sensor is unable to be placed inside the chip so it must be placed immediately outside of it during measurements inside the satellite. The temper- ature inside the chip will reach an equilibrium level near that of the outside air making this a su cient method of deciding temperature level, or probably even better, as the outside air temperature can be regulated faster if the sensor is placed there and can pick up uctuations easier. The pressure calibration measurements proved to also follow a linear trend, and a very consistent one at that, during all measurements where the sensor was subject to negative pressure. In a study where growth of the micro-organism Spirulina Platensis was monitored by pressure measurements in a closed photobioreactor, the pressure was not regu- lated until reaching 130% of the atmospheric pressure, needing only to detect a 30 kPa dierence in pressure while giving precise results. This limit also had a margin to the maximum recommended pressure level of 150% of the atmospheric pressure. [7] In relation to this, the ±80 kPa pressure dierence range available from the
44 selected pressure sensor should be more than su cient especially if measurements will show that the pressure does not increase very rapidly during the E.coli growth phase inside the chip. From the experiment protocol testing monitoring graph the pressure level did not show signs of increasing rapidly, or increasing at all. How- ever, if other bacteria should be selected for tests, such as cyanobacteria, pressure will most certainly increase during growth. Both the temperature and the pressure measurements show that the respective sensors will give equally valid results whether the supply voltage is 5 V or 3 V. However, this does not guarantee that any of the components will sustain damage that might be caused by operating at another voltage than recommended. This could be important to consider before a long experiment is initiated. The most unexpected result appeared when all the components had been assembled and the nal testing was performed. Interpreting the results from the experimental protocol led to the realization that the absorbance graph from the bacteria growth measurements was misleading, due to the fact that, not only did the absorbance curve follow the temperature curve closely in the beginning, but it also actually followed the temperature decrease at the moment which the measuring equipment was put into a room temperature environment. This is a clear indicator that a component involved in the absorbance measurements, either the photosensor, the LED or the resistors has a considerable temperature dependency. The decrease in absorbance should not occur since the concentration of bacteria cells (dead or alive) should not decrease even when the culture stops growing and starts dying o. The dependency had previously not caused any problems, at least not in the dilu- tion measurements as temperatures had been constant. However, in the end when temperatures did uctuate and measurements of temperature and absorbance were produced simultaneously and comparable side to side, the issue was revealed. This means that in the continued work, actions need to be performed in order to work around the temperature dependencies of the components, either by replacing the components that are temperature dependent, with ones without dependencies, or by calibrating the absorbance measurements after temperature measurements, in an eort to diminish the eects of the temperature dependencies as much as possible. Calibrating to temperature data will require testing on each of all the involved components to see how they individually are aected by temperature. If this can work out successfully and absorbance can be measured despite temperature uctuations, there will possibly be no need to replace any components.
45 During the growth and dilution measurements it was noted that the thinner chip yielded a voltage span that was considerably lower than measurements made on the cuvette which was a lot thicker. A thicker chip depth yields more precise results than a thinner chip in terms of measurement resolution, since the optical path length becomes larger. This is equivalent to making the opacity dierence much larger in high versus low bacteria cell concentrations for a thicker chip. However, one of the limitations of the experiment is the amount of alloted space it will have aboard the MIST-satellite, and thus the chip thickness will have to be adjusted accordingly.
6.1 Conclusion
Being one of experiments that will take place in the MIST-satellite, the MORE- BAC experiment was designed to further investigate bacteria culturing on space ights, with focus on resuscitation of freeze-dried bacteria. In this master thesis, the development of the MOREBAC measuring equipment for detecting bacteria growth and monitoring of the experiment environment was conducted. The approach for detecting bacteria growth was measuring absorbance, i.e measur- ing the rate of light passing through a sample substance containing the bacteria. Environmental control consisted of temperature and pressure measurements. For the measuring equipment, sensors, an LED, resistors, wires and a microcon- troller were connected on a breadboard. Furthermore, for the absorbance mea- surements experimental setup, the con guration consisted of the LED and the photoresistor directed toward each other, kept in place with holders and steel beams for stabilization and a mechanical iris for centralizing the light beam. Being placed in the light beam between the LED and the photoresistor, a transpar- ent bacteria culturing chip containing the test sample was used during measure- ments. Also, a custom-made culturing chip prototype made of acrylic was designed and manufactured to better t the environment of the MOREBAC experiment and the way it will be executed. The microcontroller was programmed to keep speci c electrical components active during measurements, depending on the commands it received from computer software through serial communication. Processing was the computer software used for sending commands to the microcontroller, and the Arduino software was used to program the microcontroller's responses to those commands. Tests were performed to evaluate the electrical components' behavior. The pho-
46 toresistor used for the absorbance measurements was able to dierentiate between varying bacteria cell concentrations, making it appropriate for detecting bacteria growth. However, when temperatures were varied to see how the components be- haved, a temperature dependency was discovered, the optical measurement compo- nents could only be concluded to yield reliable data for experiments with constant temperature. Even if the aim is to keep the temperature at an optimal one for bacterial growth, it will likely uctuate since the temperature regulation system will not be updating measurements at every second, but rather at larger discrete time steps, and will only kick in when reaching designated levels. The temperature and pressure sensors proved able to measure temperatures and pressures following a linear trend. The power supply of the microcontroller of 5 V was used throughout most experiments but the temperature and pressure sensors were also tested and worked just as ne for the lower supply voltage of 3 V. After having tested the sensors separately, an attempt at simulating the exper- iment as it will be performed aboard the MIST-satellite was made, except only monitoring absorbance, temperature and pressure measurements during bacterial growth. The experiment protocol was thus implemented, successfully carrying out measurements while simultaneously producing monitoring visualizations and documenting events.
6.2 Future work
For the MOREBAC experiment to be executed, there are still plenty of features surrounding the measuring equipment that need to be implemented. An imme- diate task to accomplish for absorbance measurements to become accurate is to investigate and handle the temperature dependency of the optical measurement components. Performing separate calibration with respect to temperature on the involved components could be one way of still using the same setup without chang- ing any components. Otherwise, the components can be substituted for less tem- perature dependent ones. Moreover, a life support system that can maintain the right environment for the bacteria to grow in needs to be designed. This should include pressure and tem- perature regulation, as well as a system that takes care of nutrition supply. For the pressure regulation, tubes with pinch valves that open and close to even out pressure when the pressure levels are too high, is one solution that has been con- sidered and should be implemented. In a meeting with the MIST-group at KTH,
47 it was suggested that the temperature regulation could be assisted by one of the other project-groups in charge of a neighboring experiment aboard the MIST- satellite. One of the important challenges in the future work of the project will be to manage the resuscitation of freeze-dried bacteria inside the culturing chip. Before resusci- tation, if the freeze-dried bacteria require storage at cold temperatures during the wait, the temperature sensor must be calibrated for those temperatures. It must be researched what bacteria media should be used as resuscitation activator liquid and how it should be contained before injection into the chip. Since mainly just the design of the chip has been performed, its performance it terms of heat and high pressure exposure and containment of content for longer periods of time must be tested. A factor to keep in mind when further developing the culturing chip, is the micro gravity environment in the satellite. Since the gravity pull will be quite weak, the uids and gases are prone to moving about a lot more. This induces obstacles when it comes to keeping the uids and gases in the experiment in place. One suggestion of a solution to keep uids and gases where they belong in the chip is implementing hydrophobic and hydrophilic lters, used for keeping uids and gases separate. A hydrophobic lter can be placed in the outlet channel of the chip, obstructing liquids from passing further than the culturing chamber. Similarly, a hydrophilic lter can be placed in the inlet channel, letting liquids pass on to the culturing chamber and preventing gases from going into the inlet channel. Nylon membranes can be placed next to the lters to hold them in place and protect them from wear. [18] Eventually, when the experiment will be carried out aboard the MIST-satellite in its orbit around Earth, the execution of the experiment protocol will be required to work awlessly, requiring a functioning communication between the microcon- troller and the main computer aboard the satellite. Furthermore, the experiment will consist of many parallel measurements where some are performed on a small scale time frame as in over the course of a few hours and others on a longer time interval as in, for example, over the course of weeks. As discoveries are made and the experiment becomes more well-de ned during the testing and development phase, the more important communication with the MIST team becomes. More information on exactly what they can provide in terms of alloted space, power, help with temperature regulation and general information about the launch of the satellite should be requested, as well as requirements from them what they expect in terms of measurement frequency, measurement data sizes and allowed components. Bibliography
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49 [11] J. Coonrod, What is outgassing and when does it matter?. (2010-11- 19). Retrieved from http://mwexpert.typepad.com/rog_blog/2010/11/what- is-outgassing-and-when-does-it-matter.html. [12] S. Sutton, Measurement of cell concentration in sus- pension by optical density. (August, 2006). Retrieved from http://www.microbiol.org/resources/monographswhite- papers/measurement-of-cell-concentration-in-suspension-by-optical-density/. [13] D. Barich, Thermus thermophilus. (2010-11-16). Retrieved from https://microbewiki.kenyon.edu/index.php/Thermus_thermophilus. [14] J. B. Microbiol., Growth of escherichia coli at elevated temperatures. (2005). Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/16187264. [15] J. D. van Elsas, Survival of escherichia coli in the environment: fun- damental and public health aspects. (2010-06-24). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3105702/. [16] M. T. Inc., Mcp9700 series datasheet. (2016). Retrieved from http://www.digikey.se/product-search/en?keywords=mcp9700a-e. [17] F. S. Inc., Mpx5100 series datasheet. (2010-05-13). Retrieved from http://www.digikey.se/product-search/en?keywords=MPXV5100DP. [18] V. Burt, A few guidelines for selecting lters. (July 2005). Retrieved from http://medicaldesign.com/components/few-guidelines-selecting- lters.
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