Team:SoundBio/Hardware
Hardware
The Purpose of the Bioreactor in the Bacto-Basics Project
Currently there has been much interest in the development of bacterial cellulose for a number of industrial applications, but this has been hindered by the expensive cost of BC production. This is mainly due to the low productivity of known BC producing bacterial species, and also the common usage of expensive culture media. According to one study, media costs account for 30% of total costs of BC production. In addition, there is a lack of efficient fermentation systems, and most existing systems have high operating costs and result in low yields. There is also a need for general research on optimization of the whole process, including post production modifications.Our bioreactor allows for control of the pH, oxygen level, and temperature of the growth environment, in order to provide optimal conditions for maximal cellulose production from our bacteria. As cells metabolize, their waste products can significantly affect the overall pH of the media in which they are growing in, a change that can be extremely harmful to the bacteria. This applies to temperature as well; any external temperature fluctuations need to be negated by the bioreactor so that they don’t negatively affect the bacterial cells. Additionally, as our bacteria grow and consume oxygen, the total oxygen levels in the growth environment may decrease. Since a lack of oxygen can lead to cell death, this is another crucial factor that our bioreactor must account for. Controlling, to the best of our abilities, the aforementioned variables is the fundamental purpose of our bioreactor.
We investigated multiple types of bioreactor designs, including static, wave, and bubble-column bioreactors. Our initial tests with different bioreactor types determined that bubble column bioreactors were most suitable for our project direction and eventual goals. While static bioreactors produced more uniform sheets of BC at small scale than sparged reactors, there are oxygen transfer limitations with static reactors that limit BC formation, so a sparged bubble column reactor seemed more promising long-term.
Bioreactor Design Process & Overview
Design Overview
Our bioreactor is a bubble column bioreactor. Bubble column bioreactor structures’ consist of essentially a cylindrical vessel with a sparging tube attached. Their main advantage over static culture bioreactors, is the higher heat and oxygen transfer rate due to the gas flow in a bubble column. Compared to other agitated bioreactors, bubble columns tend to be much simpler in structure, and thus are lower cost and less energy-intensive, as well as being easier to scale up. Additionally, bubble column reactors have a low shear characteristics, that are easily adjusted, which ensures that our bacterial cells are not damaged by the sparging, while effective mixing is occurring at the same time.In our design, we incorporated the traditional features of a bubble-column bioreactor by adding spargers to allow for the control of oxygen saturation. However, we also added features such as chambers and vents to allow for gas release and media/cell input, we conducted experiments to select the optimal media and growth conditions, and we incorporated a variety of sensors and other tools to monitor and functionalize our bacterial cellulose growth.
Summary of Design Process
Our plan for production of bacterial cellulose uses a bubble-column bioreactor to grow and functionalize bacterial cellulose in a co-culture of E. coli and K. rhaeticus. As proof-of-concept, we designed a custom working batch bioreactor to grow K. rhaeticus culture. Our design process was as follows:- Exploring optimization factors of BC
- Research on types of bioreactor designs
- Producing BC
- Small scale tests of bioreactor prototypes
- Culture duration and media testing
- Bioreactor design adjustments and the addition of supporting equipment
Bioreactor Design Discussion
Bioreactor Parts Overview
Our bioreactor design comprises of three main overarching categories: chambers & vents, media & culture, and sensors & functionalization. When designing our bioreactor, we want to have hardware supported both the growth of bacterial cellulose and the functionalization of bacterial cellulose. Although in wetlab we were unable to get our genetic circuits to function properly, we designed our hardware with the future goal of being successful with our genetic engineering.
Chambers and Vents
Our bioreactor is made up of several parts that together create a compatible working unit. We included an air vent with a filter to ensure that pressure does not build up within the inner chamber. It also adds another layer of protection to ensure no external contamination makes its way into the inner chamber. The inner chamber, composed of transparent polycarbonate, houses the media and the cells that produce bacterial cellulose. The inner and outer chambers are rectangular, custom-bonded plastic containers specifically designed for our bioreactor’s needs. The outer chamber will be filled with water to help maintain an optimal temperature to ensure maximal bacterial cellulose production. The outer chamber’s function is to limit the number of components in the chamber where cellulose will be growing (media, cell input tubes, etc.) and to also avoid having the sparge stones directly in contact with the cellulose to prevent disruption of the cellulose pellicle. Sparge stones embedded along the sides of the inner chamber provide the bacterial cellulose with oxygen necessary for bacterial growth.Media & Culture
Media and culture are both necessary components we considered in creating our bioreactor. The Yamanaka media and K. rhaeticus culture will be pumped into the bioreactor through their respective ports after the bioreactor has been sterilized through autoclaving. This allows for the media to enter the inner chamber without being contaminated. However, because K.rhaeticus cannot be filtered through a media filter (otherwise the cells would be filtered out), the culture will need to be pumped into the bioreactor through a sample port under a flow hood to reduce any risk of contamination.The media we have decided to use is the Yamanaka media, which was found in our research and another study to result in very high yields of cellulose, compared to mediums such as Son and CSL media, (which are also considerably more expensive to make). Prior to being pumped into our bioreactor’s inner chamber, the media components are autoclaved separately (to avoid any hazardous chemical reactions that can occur when glucose and yeast are autoclaved) and will pass through a 0.2 micron media filter to ensure sterility. Yamanaka efficiently provides nutrients to the K. rhaeticus growing inside the bioreactor. E. coli will also be introduced via the sprinkler system during the growth period of K. rhaeticus to guarantee that proteins are successfully embedded into the cellulose.
Sensors & Functionalization
pH sensor: We used an analog pH sensor for measuring the pH of the media. In combination with pH adjustments through the upper tubes, the sensor will allow us to maintain the growth environment at an optimum pH level for K. rhaeticus growth. For example, the pH sensor readings will help us readjust the acidity if any possible pH changes occur. We will use a PID controller with the sensor to control the acidity within an acceptable range.Dissolved oxygen sensor: We used for measuring the dissolved oxygen level in the bioreactor. Controlling the dissolved oxygen levels is also important for achieving optimal cellulose production.
Temperature sensor and heating system: We used for measuring the temperature of the inner container to ensure the temperature of the bioreactor remains constant. It will be placed in the water in the outer layer of the bioreactor in order to minimize the number of probes disrupting the production of BC in the growth chamber, and the temperature of the whole system will be controlled by an incubator or, in the future, a temperature blanket using a PID system.
Functionalization: We are using an LED array constructed from Raspberry Pi controlled LED strips, which are made from flexible, waterproof, black PCB material. This is used in the functionalization of the cellulose. Referencing light intensity and output tables, we aim to be able to functionalize the cellulose precisely and accurately through light, with gradientation possible through PWM control of the array. This LED array is attached to the transparent lid of our bioreactor, after autoclaving, to allow light to shine onto the growing BC with relative ease.
Bioreactor Growth Conditions
Culture Duration
Culture duration is the length of time for bacterial cellulose to reach maximal production. Typically in bacterial cell cultures, short incubation time would result in minimal growth whereas an excessively long incubation would result in overcrowding and subsequently less bacterial growth. To determine the optimal incubation time for our K. rhaeticus culture, we conducted tests in our lab. We seeded 21 conical tubes with our culture, and incubated them at 30 C. Starting from the 4 day mark since seeding, we harvested 3 of the tubes every 2 days and recorded the mass of bacterial cellulose in each sample. This was conducted over a period of 14 days. We observed increased bacterial growth up until ten days, and it subsequently began to decrease. We concluded that the optimal duration for bacterial cellulose growth was ten days.
Carbon Source & Media Selection
The role of media in a culture is to provide nutrients for cells to grow. We conducted several tests to determine which media resulted in the most bacterial cellulose production. We tested two media types, Yamanaka media and Hestrin-Schramm media, and we found that Yamanaka was able to produce a full sheet of BC in a week while the Hestrin-Schramm media needed one more week to produce the same amount of BC. Additionally, to determine if our bacteria had a preference for the carbon source used in the media, we tested mannitol, sucrose, and glucose in Hestrin Schramm media since these three carbon sources tended to be the most effective. In our experiment, 36ml of the media was added to 4ml of K. rhaeticus cell stock in a 50 ml conical tube, with three tubes per carbon source. The cultures were then incubated at 30 C for 7 days. After washing and drying the bacterial cellulose from each tube, our data indicated that glucose and mannitol were the best carbon sources for maximum cellulose production, as tubes with those carbon sources had an average BC mass of .47 grams.
Culture Ratio
A culture ratio is essentially the proportion of cells to media in a culture. It is important for this ratio to be optimized based on the cell species and media type, because it can vary greatly. A certain cell species will consume nutrients from a medium faster than another, so a determined ratio will help ensure that there are enough media nutrients for the cells in a given culture for a period of time. If not determined, an inaccurate culture ratio can cause cell death because there may be too many cells for a given amount of media.To determine the optimal culture ratio, we seeded 6 200ml pyrex bottles, with a total of 100ml of solution in each bottle. We tested a different culture ratio in each bottle, using cell to media ratios of 1:5, 1:10, 1:20, 1:50, 1:100, and 1:150. Unfortunately, these results were inconclusive, even after a second attempt because the bacterial cellulose was generally too thin to be dried accurately. Since our prior experiments unrelated to culture ratio had relatively high cellulose production at a 1:10 culture ratio, this was the ratio we decided to continue to use.
Another variable to consider is the surface area to culture ratio of the vessel (whether or not a vessel with a small surface area will produce more/less/same amount of BC as a vessel with a bigger surface area). We decided to conduct an experiment with a constant of the cell ratio, but tested the surface area to culture ratio, and settled on a 1:2 ratio based on the data we collected.
Oxygen
Oxygen is useful for optimal growth. Allowing enough oxygen to keep the culture healthy was one of the primary reasons we chose a bubble-column bioreactor. We used a DO probe to sense when oxygen was low, and used a sparging mechanism to oxygenate the media. We programmed the dissolved oxygen probe to a computer using Raspberry Pi and use that to digitally monitor the oxygen levels at our convenience. The dissolved oxygen probe will tell us how much oxygen has been absorbed by the culture, which will inform us how well the k.rhaeticus is producing BC.Temperature
In bacterial culture, temperature increases enzyme activity that allows bacterial cellulose to be produced and the culture to prosper. However, high temperatures can result in loss of this enzyme activity and the denaturation of aiding proteins, whereas low temperatures can result in lower enzyme activity. As a result, it is important to make sure the temperature is high enough to stimulate a large amount of enzyme activity without negatively impacting proteins. In our bioreactor, we are filling the outer chamber with water at 30 degrees Celsius so that it can evenly distribute the heat throughout the bacterial cellulose environment within the lower chamber. Research has shown that the optimal growing temperature for bacterial cellulose is 30 degrees celsius, and that has been corroborated through all of our BC experiments. We are able to maintain optimal temperature by programming out temperature probe to provide accurate temperature readings and adjusting our incubator temperature accordingly.pH
Optimal pH is necessary in a bacterial cellulose culture. Because bacteria only grows under specific conditions, pH has to be controlled in order to ensure the bacteria is not negatively impacted. If pH is too acidic or too basic, the bacteria’s metabolism will be paused and its catalytic enzymes will be ineffective. Therefore, we will use a pH sensor to ensure optimal growth conditions for the bacterial cellulose. Additionally, the pH of the media used is also a specific number (pH of 5) because it has been tested to be beneficial to the production of bacterial cellulose. Our pH sensor will be one of the only external objects in the inner chamber where pH will be closely monitored. By programming it with Raspberry Pi, we are able to effectively and efficiently keep pH maintained under favorable conditions.Lab Notebooks/Experiments
Protocols
Assembling Our Bioreactor: Our optimal bioreactor design for growing BC as it increases the oxygen availability for the K. rhaeticus. It also includes the sensors, allowing us to control and maintain a sustainable environment.Creating Yamanaka Agar and Streaking w/ K. rhaeticus: Making agar with yamanaka media to use in K. rhaeticus petri dishes, to isolate individual colonies of K. rhaeticus.
K. rhaeticus HS Media and Culturing: Hestrin- Schramm media has been found to be one of two preferable medium for BC production. Contains carbon source, enriched nitrogen source, and a small amount of citric acid. There are two options for culturing: in plates or in a liquid medium.
Starting a K. rhaeticus Stock: Using the cryostock to start a new batch of cultures that we can use to seed experiments.
Media Recipes
CSL MediaSon Media
Yamanaka Media
Zhou Media
Lab Notebooks
Agitation Tests Procedure and ResultsThese notebooks contain the procedure and results from an experiment that tested the effect of agitation on cultures and their BC yield and quality, mimicking a wave bioreactor.
Bioreactor Prototype Testing
This notebook documents an experiment which tested the first iteration of our bioreactor that aimed to prevent the clumping of cellulose around our sparge stones, an issue we experienced in previous designs.
Bioreactor Type/Material Testing
This notebook documents an experiment that aimed to test the effect of the type of bioreactor (airlift, static, and wave) and the material of the container (pyrex, polystyrene, and polypropylene) and their effects on BC yield and quality.
Carbon Source Testing
This notebook contains protocol and results for an experiment that tested whether glucose, mannitol, or sucrose proved to be a more effective carbon source for producing high BC yields.
Culture Duration Test
This notebook includes a protocol and data for an experiment to determine the optimal incubation period for our cultures.
Growth with Pre-Existing BC Protocol and Growth Tests with Starting BC Sheet
This protocol is for testing BC growth with a preexisting sheet present, to see what the effects of the preexisting cellulose are on the yield and quality of newly produced bacterial cellulose. The notebook includes some of our preliminary tests of BC growth in an airlift bioreactor. It also places a focus on whether placement of BC affects amount of BC.
Preparation for Testing in Prototype Bioreactor
This notebook includes the steps and procedures that need to be followed to prepare a culture for for testing in our final prototype bioreactor.
Producing our Media - Yamanaka and Hestrin Schramm
These notebooks describe the procedures we used to produce the above mentioned media.
Quantification of BC Production
These procedures aim to quantify the mass of bacterial cellulose produced from our cultures. The second notebook was modified after the first procedure appeared to be unreliable.
Seed Density Testing
This notebook includes a protocol and data for an experiment that attempted to determine the ratio of cell stock to media that produced the highest BC yield.
Sensor & R Pi Setup
This notebook includes our progress in programming our various sensors.
Sparging Tests Procedure and Results
This notebook includes our progress in programming our various sensors.
Testing the Effect of the Shape of Growth Vessel on BC Growth
This notebook includes the testing of vessel shape and its effect on BC growth with a focus on rectangular vessels and circular vessels.
Testing Yamanaka Media vs HS
This notebook describes an experiment that tested the efficacy of Yamanaka media compared to Hestrin Schramm media in K. rhaeticus cultures for production of high BC yields.
Troubleshooting Airlift Reactor with Silicone Protocol and Results
These notebooks contain protocols and results from an experiment that aimed to solve an issue of cellulose growth around sparge stones with the addition of a silicone layer around the sparge stone to deter cellulose growth.