Difference between revisions of "Team:NUS Singapore-A/Hardware/Futuristic Bioreactor"

(Created page with "{{NUS_Singapore-A}} {{:Team:NUS_Singapore-A/Templates/Style}} <html> <head> <link href="https://fonts.googleapis.com/css?family=Montserrat:300,400,600" rel="stylesheet"> <lin...")
 
Line 22: Line 22:
 
<img src="https://static.igem.org/mediawiki/2018/e/e5/T--NUS_Singapore-A--Hardware_header_C.png" class="header">
 
<img src="https://static.igem.org/mediawiki/2018/e/e5/T--NUS_Singapore-A--Hardware_header_C.png" class="header">
  
<h1>Fermentation Chamber (INNOVATION)</h1>
+
<h1>Future design considerations</h1>
<br>
+
<p>  
<looping gif></looping>
+
Goal: Improve production efficiency
<br>
+
</p>
<h2>Function</h2>
+
<p>
<br>
+
<h2>Achieved by</h2>
<p>The fermentation chamber contains the bacterial culture. It also comes with a cover designed to include a means of illuminating the chamber’s contents. </p>
+
<ol>
<br>
+
<li>Grow E. Coli at high cell densities</li>
 +
<li>Avoid substrate inhibition</li>
 +
<li>Avoid accumulation of inhibitory compounds</li>
 +
<li>Avoid selection of auxotrophic mutants</li>
 +
<li>Reduce downtime</li>
 +
</ol>
 +
</p>
 +
<p>
 +
<h2>Design considerations</h2>
 +
<ol>
 +
<li>Process Type</li>
 +
<li>Feed Profile</li>
 +
<li>Nutrient Delivery</li>
 +
<li>Lighting Configuration</li>
 +
<li>Sensing Cell Stress</li>
 +
<li>Biphasic Media</li>
 +
</ol>
 +
</p>  
 +
<h3> Process Type </h3>
 +
<p>
 +
Fed-batch processes rare popular because they allow limiting substrates to maintained in constantly low concentrations and keep the cells at low growth rate.
 +
This allows high cell density as the nutrients are constantly being replaced.
 +
Meanwhile, the low substrate concentration prevents substrate inhibition, and the selection of auxotrophic mutants.
 +
Finally, fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles.
 +
</p>
 +
<p>
 +
Continuous production can also address these challenges.
 +
However, it presents many operational challenges at industrial scale, as it requires tightly controlled conditions and robust monitoring methods.
 +
There may also be scheduling challenges as the downstream operations cannot always be operated continuously.
 +
In addition, the long operation requires a genetically stable host system, and there is also a higher risk of contamination.
 +
</p>
  
<h2>Design</h2>
+
<h3>Feed Profile</h3>
<br>
+
<p>
<p>We selected components of the fermentation chamber based on whether they were easily obtainable and modifiable. This was because it was difficult to fabricate a cylindrical, watertight chamber using conventional methods of prototyping such as laser cutting and 3D printing. Rather than spend time attempting to manufacture a suitable container from scratch, we looked to modifying existing commercial, easily obtainable products. This also makes it easier for others to make their own bioreactor based on our work.</p>
+
Based on our interviews with Dr. Nic Lindley, we learnt that using xylose as a substrate instead of glucose makes E.Coli, grow slower but more sustainably.
<br>
+
This is because glucose metabolism produces acetic acid which inhibits growth.
 +
In addition, slowing down growth frees up cellular resources such as ribosomes for producing other proteins, thus offsetting the protein burden.
 +
</p>
 +
<p>
 +
Another tip we learned was to use nitrogen sources as the limiting growth factor instead of sugar.
 +
Low sugar concentrations select for cells that grow quickly at the expense of bioproduction.
 +
Having sufficient sugar will ensure the cells’ growth requirements are satisfied and that a significant portion of the energy budget can be used for production.
 +
</p>
  
<h3>Container</h3>
+
<h3> Nutrient Delivery</h3>
<br>
+
</p>
<p>We decided on a suitable working volume for our bioreactor based on literature review<sup>[1]</sup>. Possible working volumes suitable for the laboratory ranged from 0.5 L  to 2 L. After discussion, we decided that 0.5 L would be a manageable volume. We hypothesized that this volume was sufficient for us to demonstrate proof-of-concept. It is also more portable.</p>
+
A simple design for a bioreactor would have nutrients delivered from the top and oxygen from the bottom.
<br>
+
However, the large size of industrial bioreactors means that significant heterogeneity can occur within the reactor due to long mixing time.
 +
As we learnt from our interview with Dr. Nic Lindley, such a bioreactor is polarized into a nutrient-rich, anoxic zone at the top and a nutrient-poor, oxygenated zone at the bottom.
 +
This is the result of bacteria consuming all the oxygen or food at the delivery point.
 +
Performance of the main metabolic pathway becomes difficult to predict and other unwanted pathways may be activated.
 +
</p>
 +
<p>
 +
It has demonstrated that generation of turbulent flow can improve E.Coli growth by up to five times compared to still water controls<sup>[1]</sup>.  
 +
We intend to keep the tank-to-impeller diameter ratio between 1.6 to 2 times to prevent the formation of caverns.  
 +
In addition, we will use a close clearance helical impeller that sweeps a large proportion of the tank volume with a Rushton turbine that ensures good gas dispersion<sup>[3]</sup>.
 +
</p>
  
 +
<h3>Lighting Configuration</h3>
 +
<p>
 +
Since the bacterial system is controlled by light, it is important that light penetration is adequate throughout the tank.
 +
We will thus borrow ideas from photobioreactors which are normally used for algae;
 +
internally illuminated photobioreactors have been proposed to increase efficiency by decreasing the path length of light<sup>[7[</sup>.
 +
Since we already have a stirrer in the center of the reactor adding lights internally will mean installing the lights on the impellers themselves.
 +
Unlike a photobioreactor, we do not want light from outside to enter the reactor.
 +
In addition, we want minimize the loss of light energy due to the reactor wall.
 +
Our reactor wall will thus be opaque and reflective to maximize the illumination within the reactor.
 +
</p>
  
<p>A bail jar from IKEA was repurposed for this. Discarding the original glass cover, we retained the rubber ring and two-part wire clasp.</p>
+
<h3>Sensing Cell Stress</h3>
<br>
+
<p>
 +
In the case of our system, the control objectives are to prioritize maximizing process yield while maintaining a constant, low oxygen profile and by-product formation.
 +
One development we have in mind is the use of the spinach reporter system for measuring cell stress.
 +
This was demonstrated by the wet lab team.
 +
As of now, cell stress is normally measured indirectly by the looking medium properties such as dissolved oxygen, pH and conductivity.
 +
Using spinach, we can use measure fluorescence to directly understand the level of stress the cells experience.
 +
This will significantly reduce uncertainty and lead to better control strategies.
 +
</p>
  
<h3>Cover</h3>
+
<h3>Biphasic Media</h3>
<br>
+
<p>
<p>We opted not to use the original cover because we knew we would have to perforate it to accommodate tubing. Drilling would generate many points of failure. For example, the glass cover might shatter from the stress, it is challenging to control the accuracy of our drilling, and drilling may not be able to provide the required precision.</p>
+
Biphasic media systems have been shown to offer several advantages in bioprocessing; the product is moved into the organic phase and reduces toxicity and thus increases yield<sup>[4]</sup>. At the same time, the separation of the product from the cells makes recovery easier.  
<br>
+
The organic phase is a hydrocarbon solvent (n-decane, n-hexadecane) with an octanol:water partition coefficient (log P) above 4.
 
+
This a commonly accepted requisite for a good tolerance by whole microbial cells <sup>[4]</sup>.
<p>We thus retrofitted our own lasercut Plexiglass cover. This allowed us greater flexibility to explore potential sensing modules and components we intended to add to our system. Considered, but eventually discarded, were modules like a pH meter, as we decided to focus on other metrics to indicate cell stress (See <a href="#">Design: Stress Reporter</a>). Eventually, we decided to have 4 small holes, through which acrylic/glass tubes with outer diameters of 6 mm should be inserted, and 2 large holes, to each fit a test tube (Figure 1).</p>
+
Since luteolin has been shown to be readily soluble in alkanes <sup>[5]</sup>, we believe that a vegetable oil solvent can provide adequate performance while eliminating concerns of toxicity<sup>[6]</sup>.
<br>
+
</p>
<p>For the acrylic tubes, 2 tubes would be connected to a peristaltic pump, one to an air pump, and the last one to a length of silicone tubing so that new media can be introduced. The test tubes act as containers for LEDs, allowing them to suffuse the culture with light (Figure 2).</p>
+
<h3> Futuristic Bioreactor </h3>
<br>
+
<p> Our envisioned futuristic bioreactor is designed below to include the features discussed above.  
<figure class="figures2">
+
<h2> References</h2>
<img src="#">
+
[1] Hondzo, M., & Al‐Homoud, A. (2007). Model development and verification for mass transport to Escherichia coli cells in a turbulent flow. Water resources research, 43(8).<br><br>
<figcaption>Figure 1. Top view of lasercut cover, showing the locations of apertures for acrylic tubes and test tubes.</figcaption>
+
[2] Mears, L., Stocks, S. M., Sin, G., & Gernaey, K. V. (2017). A review of control strategies for manipulating the feed rate in fed-batch fermentation processes. Journal of biotechnology, 245, 34-46. <br><br>
 
+
[3] Cabaret, F., Fradette, L., & Tanguy, P. A. (2008). Gas–liquid mass transfer in unbaffled dual-impeller mixers. Chemical Engineering Science, 63(6), 1636-1647.<br><br>
<img src="#">
+
[4] Ratledge, C., & Kristiansen, B. (Eds.). (2006). Basic biotechnology. Cambridge University Press.<br><br>
<figcaption>Figure 2. Section view of fermentation chamber, showing how test tubes can provide an illumination solution. Also included is an example of how an acrylic tube may be inserted into the cover. Tubing and wiring have been omitted for clarity.</figcaption>
+
[5] Peng, B., Zi, J., & Yan, W. (2006). Measurement and correlation of solubilities of luteolin in organic solvents at different temperatures. Journal of Chemical & Engineering Data, 51(6), 2038-2040.<br><br>
</figure>
+
[6] Bicas, J. L., Fontanille, P., Pastore, G. M., & Larroche, C. (2010). A bioprocess for the production of high concentrations of R-(+)-α-terpineol from R-(+)-limonene. Process biochemistry, 45(4), 481-486.<br><br>
<br>
+
[7] Pegallapati, A. K., Arudchelvam, Y., & Nirmalakhandan, N. (2012). Energy-efficient photobioreactor configuration for algal biomass production. Bioresource technology, 126, 266-273.
 
+
<h3>Stirring</h3>
+
<br>
+
<p>The ambient temperature of where our bioreactor would be located was too cold to be conducive for growing bacteria. Another problem we faced was the magnetic stirrer limiting the depth to which the test tubes could be sunken and thus the light penetration. We had a eureka moment when we realized that both problems could be solved by placing Light Wait into a shaking incubator as shown in Light Wait: Product Demonstration.</p>
+
<br>
+
<hr>
+
<br>
+
<h2>Construction</h2>
+
<br>
+
<p>Do you like cookies? Do you like jars? Your answer doesn’t matter. Make our cover anyway! Batteries not included.</p>
+
<br>
+
 
+
<h3>Bill of Materials</h3>
+
<br>
+
<ul>
+
<li>180 x 100 x 5 mm acrylic sheet x 1</li>
+
<li>Arduino Uno  x 1</li>
+
<li>472 nm LED x 16</li>
+
<li>LED driver x 1</li>
+
<li>2 kΩ resistor x 1</li>
+
<li>0.1 uF ceramic capacitor x 1</li>
+
<li>4.7uF electrolytic capacitor x 1</li>
+
<li>28-pin IC socket x 1</li>
+
<li>Test tube x 2</li>
+
<li>AC Adapter x 1</li>
+
</ul>
+
<br>
+
 
+
<h3>Structural Assembly</h3>
+
<br>
+
<figure class="figures2">
+
<img src="#">
+
<figcaption>Figure 3. Fermentation Chamber assembly.</figcaption>
+
</figure>
+
<br>
+
 
+
<p>Files for lasercutting are available in SLDPRT format so that you may easily modify its dimensions to fit your jar (please find your own). You may download them all as a ZIP file <a href="#">here</a>.</p>
+
 
+
<br>
+
<hr>
+
<br>
+
<div style="text-align: left;">
+
<sup>[1]</sup>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876151/
+
</div>
+
<br>
+
  
 
</div></div>
 
</div></div>

Revision as of 13:33, 17 October 2018

CONNECT WITH US

Future design considerations

Goal: Improve production efficiency

Achieved by

  1. Grow E. Coli at high cell densities
  2. Avoid substrate inhibition
  3. Avoid accumulation of inhibitory compounds
  4. Avoid selection of auxotrophic mutants
  5. Reduce downtime

Design considerations

  1. Process Type
  2. Feed Profile
  3. Nutrient Delivery
  4. Lighting Configuration
  5. Sensing Cell Stress
  6. Biphasic Media

Process Type

Fed-batch processes rare popular because they allow limiting substrates to maintained in constantly low concentrations and keep the cells at low growth rate. This allows high cell density as the nutrients are constantly being replaced. Meanwhile, the low substrate concentration prevents substrate inhibition, and the selection of auxotrophic mutants. Finally, fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles.

Continuous production can also address these challenges. However, it presents many operational challenges at industrial scale, as it requires tightly controlled conditions and robust monitoring methods. There may also be scheduling challenges as the downstream operations cannot always be operated continuously. In addition, the long operation requires a genetically stable host system, and there is also a higher risk of contamination.

Feed Profile

Based on our interviews with Dr. Nic Lindley, we learnt that using xylose as a substrate instead of glucose makes E.Coli, grow slower but more sustainably. This is because glucose metabolism produces acetic acid which inhibits growth. In addition, slowing down growth frees up cellular resources such as ribosomes for producing other proteins, thus offsetting the protein burden.

Another tip we learned was to use nitrogen sources as the limiting growth factor instead of sugar. Low sugar concentrations select for cells that grow quickly at the expense of bioproduction. Having sufficient sugar will ensure the cells’ growth requirements are satisfied and that a significant portion of the energy budget can be used for production.

Nutrient Delivery

A simple design for a bioreactor would have nutrients delivered from the top and oxygen from the bottom. However, the large size of industrial bioreactors means that significant heterogeneity can occur within the reactor due to long mixing time. As we learnt from our interview with Dr. Nic Lindley, such a bioreactor is polarized into a nutrient-rich, anoxic zone at the top and a nutrient-poor, oxygenated zone at the bottom. This is the result of bacteria consuming all the oxygen or food at the delivery point. Performance of the main metabolic pathway becomes difficult to predict and other unwanted pathways may be activated.

It has demonstrated that generation of turbulent flow can improve E.Coli growth by up to five times compared to still water controls[1]. We intend to keep the tank-to-impeller diameter ratio between 1.6 to 2 times to prevent the formation of caverns. In addition, we will use a close clearance helical impeller that sweeps a large proportion of the tank volume with a Rushton turbine that ensures good gas dispersion[3].

Lighting Configuration

Since the bacterial system is controlled by light, it is important that light penetration is adequate throughout the tank. We will thus borrow ideas from photobioreactors which are normally used for algae; internally illuminated photobioreactors have been proposed to increase efficiency by decreasing the path length of light[7[. Since we already have a stirrer in the center of the reactor adding lights internally will mean installing the lights on the impellers themselves. Unlike a photobioreactor, we do not want light from outside to enter the reactor. In addition, we want minimize the loss of light energy due to the reactor wall. Our reactor wall will thus be opaque and reflective to maximize the illumination within the reactor.

Sensing Cell Stress

In the case of our system, the control objectives are to prioritize maximizing process yield while maintaining a constant, low oxygen profile and by-product formation. One development we have in mind is the use of the spinach reporter system for measuring cell stress. This was demonstrated by the wet lab team. As of now, cell stress is normally measured indirectly by the looking medium properties such as dissolved oxygen, pH and conductivity. Using spinach, we can use measure fluorescence to directly understand the level of stress the cells experience. This will significantly reduce uncertainty and lead to better control strategies.

Biphasic Media

Biphasic media systems have been shown to offer several advantages in bioprocessing; the product is moved into the organic phase and reduces toxicity and thus increases yield[4]. At the same time, the separation of the product from the cells makes recovery easier. The organic phase is a hydrocarbon solvent (n-decane, n-hexadecane) with an octanol:water partition coefficient (log P) above 4. This a commonly accepted requisite for a good tolerance by whole microbial cells [4]. Since luteolin has been shown to be readily soluble in alkanes [5], we believe that a vegetable oil solvent can provide adequate performance while eliminating concerns of toxicity[6].

Futuristic Bioreactor

Our envisioned futuristic bioreactor is designed below to include the features discussed above.

References

[1] Hondzo, M., & Al‐Homoud, A. (2007). Model development and verification for mass transport to Escherichia coli cells in a turbulent flow. Water resources research, 43(8).

[2] Mears, L., Stocks, S. M., Sin, G., & Gernaey, K. V. (2017). A review of control strategies for manipulating the feed rate in fed-batch fermentation processes. Journal of biotechnology, 245, 34-46.

[3] Cabaret, F., Fradette, L., & Tanguy, P. A. (2008). Gas–liquid mass transfer in unbaffled dual-impeller mixers. Chemical Engineering Science, 63(6), 1636-1647.

[4] Ratledge, C., & Kristiansen, B. (Eds.). (2006). Basic biotechnology. Cambridge University Press.

[5] Peng, B., Zi, J., & Yan, W. (2006). Measurement and correlation of solubilities of luteolin in organic solvents at different temperatures. Journal of Chemical & Engineering Data, 51(6), 2038-2040.

[6] Bicas, J. L., Fontanille, P., Pastore, G. M., & Larroche, C. (2010). A bioprocess for the production of high concentrations of R-(+)-α-terpineol from R-(+)-limonene. Process biochemistry, 45(4), 481-486.

[7] Pegallapati, A. K., Arudchelvam, Y., & Nirmalakhandan, N. (2012). Energy-efficient photobioreactor configuration for algal biomass production. Bioresource technology, 126, 266-273.