Team:SDU-Denmark/Hardware
Hardware
Abstract
This is the process of how we made our rotor for our small-scale bioreactor
experiments, tests, results and the considerations we made along the process.
Introduction
Our final plan is to implement our plasmids into Rhodovulum Sulfidophilum, a marine photosynthetic purple bacterium. R. Sulfidophilum can grow under photoautotrophic and photoheterotrophic conditions, making it a very sustainable choice for an organism to use for this production, potentially producing this medicament from aged seawater and sunlight/far-red light.This means that the constant renewing of the growth medium is not necessary, and therefore do not require much to keep going for a long time, therefore making it more sustainable than other organisms.
R. Sulfidophilum has earlier been used for heterologous production of spider silk with success by Foong et al [1]. Furthermore, they transferred the entire system of enzymes via conjugation using pCF1010-derived plasmids and E. coli S17-1 as a donor strain. With this as an inspiration, we wanted to see if it was possible to use this organism to produce psilocybin. However, before we can test this, we need to optimize the growth in a bioreactor-conditions to set-up pilot scale experiments. To do this, we set up a small-scale experiment, where we used a 3D printed rotor model to get an even and consistent movement of the medium to mimic bioreactor conditions. [2] We did not have access to real bioreactors, so we made our own small pilot reactor. This was not only our option, but even if access to a real scale bioreactor was possible, one would have been too expensive to use for this project.
3D modelling and assembly of the rotor
No member on the team had any prior experience with 3D modelling, so we assembled a small team to research and design this. First, we found out that we needed a cad program to design the 3D model of our rotor. The model used in our small experiments was made in the free web-based cad-program, www.tinkercad.com.
To print the model, we needed to know which 3D printers were available at the university. The model was printed on an Ultimaker 2+ printer which was available through the University of Southern Denmark. So, to go from an idea to a final print, we needed to use the Ultimaker Cura software to finalize the design and select the correct settings for the print.
Two designs were made and printed in PLA plastic, but only one had been assembled to completion. The assembly was the combination of the two parts printed. A magnet was placed in the centre of the bottom piece, and tiny glass beads were placed in the two side chambers in the bottom portion. After that, the two pieces were glued together.
The first wave of experiments
To do these experiments, we took inspiration from a team who used R. Sulfidophilum to produce Bioplastic. [3] A chain of initiation experiments was carried out with the first rotor model. The goal of these was to see if the rotor could be sterilized. However, this was not successful, as all the experiments ended with growth of contamination in the medium. To combat this contamination problem, the rotor was washed for 30 minutes with 96% ethanol before the initiation of the experiments. However, these all ended in contamination.
First protocol
- Wash the rotor with ethanol (96%).
- Sterilization of work surface.
- Sterilize the surface of a 250ml conical flask using fire.
- Pour about 130 ml marine broth growth medium into the conical flask. *prior to the pouring, place the container in which the medium is kept, over the flame.
- Place rotor into the flask.
- Place a breath easy membrane over the opening of the flask.
- Place the flask with medium and rotor onto a magnet plate which is placed in a 30°C incubator, with a red LED light with light at about 730 nm wavelength. *
- Check the flask after 12 to 24 hours. If no growth is visible then the rotor is sterile, if the medium is cloudy then the medium has been contaminated.
* 30°C and Far-red light is because of R. Sulf, you can change these values to your bacteria e.g. 37°C without Far-red light for E. coli.
Conclusion of the first wave
Since the first wave of initiation experiments all ended in contamination, we thought the base of the problem was the duration time of the ethanol wash.
The second wave of experiments
To eliminate the contamination problem, our first instinct was to emerge the rotor in ethanol for different amounts of time. Therefore, we took a 100 ml blue cap flask and filled it with 96% ethanol. The first time this method was used, the rotor was emerged for. The second time for 2 hours, the third time for 24 hours.
Second protocol
- Emerge the rotor in 96% ethanol up to 24 hours.
- Sterilization of work surface.
- sterilize the surface of a 250ml conical flask using fire.
- Pour about 130 ml marine broth growth medium into the conical flask. *prior to the pouring, place the container in which the medium is kept, over the flame.
- Place rotor into the flask.
- Place a breath easy membrane over the opening of the flask.
- Place the flask with medium and rotor onto a magnet plate which is placed in a 30-degree Celsius incubator, with a red LED light with light at about 730 nm wavelength.*
- Check the flask after 12 to 24 hours. If no growth is visible then the rotor is sterile, if the medium is cloudy then the medium has been contaminated.
* 30°C and Far-red light is because of R. Sulf, you can change these values to your bacteria e.g. 37°C without Far-red light for E. coli.
Conclusion of the second wave
The second wave of experiments was still affected by contaminations. Here we considered a few options; one was to coat the rotor in epoxy, the other was to get a higher quality print, and the last was to get one made in a sturdier material. But the one we went with was to get our rotor coated in epoxy. As we were waiting for the epoxy to dry, a master’s student from the RUMM unit at SDU, Emilie-Martine Sixhøj Jensen, offered to print a new rotor model on a Prusa mini +.
Third wave of experiments
With the rotor coated in epoxy, and a new rotor, provided by Emilie-Martine. The third wave of experiments was tested. Like all the others, the first was carried out, but the second was carried out in a measuring cylinder. Together with this, the protocol got updated slightly to see if that had any influence over the outcome of the experiments.
Third protocol
- Emerge the rotor in 96% ethanol up to 24 hours.
- Sterilization of work surface.
- Burn the top and inside of a 100ml measure cylinder.
- Pour about 55 ml marine broth growth medium into the cylinder. *prior to the pouring, place the container in which the medium is kept over the flame.
- Place rotor into the cylinder.
- Place a breath easy membrane over the opening of the cylinder.
- Place the flask with medium and rotor onto a magnet plate which is placed in a 30°C incubator, with a red LED light with light at about 730 nm wavelength.
- Check the flask after 12 to 24 hours. If no growth is visible then the rotor is sterile, if the medium is cloudy then the medium has been contaminated.
* 30°C and Far-red light is because of R. Sulf, you can change these values to your bacteria e.g. 37°C without Far-red light for E. coli.
Conclusion of the third wave
With the protocol updated, the results finally started to look better. One morning the medium was transparent, and there were no signs of any contamination. Preperations of the next phase of experiments were engaged, here the addition of the addition of R. Sulfidophilum. During preparations, which took approximately 16-24h, the medium showed signs of growth, meaning contamination. So, then the time came to add the R. Sulfidophilum, the medium had started to show signs of growth due to contamination. The reason why the growth of contamination first began after 24 hours is still unknown. Still, we theorize that is due to ethanol leftover on the rotor and the addition of less marine broth, thereby making the ethanol concentration in the medium-high enough to slow the growth of the contaminating bacteria. To confirm that the cloudiness of the medium was due to bacterial contamination, we used microscopy to see if any bacteria could be seen in the medium. As seen in the microscopy picture below, a diverse group of bacteria caused the contamination.
Why we only used ethanol as sterilizer
Many ideas were recommended by members of the team and our supervisors. But we ended up still using ethanol as we knew the rotor could handle the exposure to ethanol and work theoretically. Some of the other things that could have worked, for example, UV, antibiotic cocktail, chlorine, or autoclaving, was not tested but should be considered for the future. Some arguments were made for why we did not try any of the other sterilization methods. For UV, we were uncertain if the UV light could get into the irregularities in the structure of the rotor, where we believe the contaminants may be. For autoclaving, we were unsure for the rotor's structural integrity could withstand the pressure of the autoclave, due to the thin walls in our design. In theory the PLA should withstand the temperature of the autoclave, since the melting point for PLA is around 170 degrees Celsius. But that was a risk we did not want to take.
Considerations for the future
For the future, we would recommend putting all the other sterilisation methods to the test to see if they are better than the emersion in ethanol. Perhaps another material than PLA plastic could also be a viable solution. Maybe plastic is not the best material for these experiments; maybe a metal rotor could have been a better choice, however we are aware that this is not as available to all as 3d printers are now a days, the accessibility of 3d printing was why we chose this route so other teams with a limited budget could achieve the same thing.
Other considerations
To see if it was the rotor or just the way we handled the preparation of the experiments, we used a control flask where we heated it the same way over a Bunsen burner and added the same amount of medium and a breath easy membrane over. The only difference between the two flasks was the rotor. There were no signs of growth in all controls, while in the ones with the rotor, there was always unwanted bacterial growth. Furthermore, only one of the designs was tested because we deemed it unnecessary to begin testing the second design until we had a usable result from the first design.
References
[2] Clapp, K. and Lindskog, E. Single-Use Bioreactor Mixing: How to meet familiar requirements with novel technologies. Cell Culture Dish Inc. October 20, 2013. Available from: https://cellculturedish.com/single-use-bioreactor-mixing-how-to-meet-familiar-requirements-with-novel-technologies/
[3] Carlozzi P, Touloupakis E. Bioplastic production by feeding the marine Rhodovulum sulfidophilum DSM-1374 with four different carbon sources under batch, fed-batch and semi-continuous growth regimes. N Biotechnol. 2021 May 25;62:10-17. doi: 10.1016/j.nbt.2020.12.002. Epub 2020 Dec 14. PMID: 33333263.
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