Week 1 HW: Final Project Ideas

1. What you would like to biomanufacture and why?

I would like to be able to biomanufacture essential oils, particularly the aromatic compounds responsible for the scents and properties of different oils. I have always enjoyed the scents of essential oils and have been interested in their many applications, including fragrances, personal care products, and other consumer products.

I am particularly interested in exploring whether compounds that are normally obtained from plants could instead be produced using other biological systems. This makes essential oils an interesting target for biomanufacturing because it could connect my interest in natural products with synthetic biology and fermentation.

My motivation for this project is primarily personal rather than utilitarian. I am interested in essential oils and how they are produced, and I think it would be fascinating to explore how biological systems could be engineered to produce these compounds. Ultimately, I would like to develop a way to produce useful and appealing aromatic compounds through a controlled biological manufacturing process.

2. What living system would you use?

I would like to use either a fast growing plant species, such as Nicotiana, or a microbial system such as bacteria or yeast. Each approach has advantages and challenges that I would like to explore.

Using a plant as the manufacturing chassis would allow the plant’s natural metabolic pathways to produce the compounds that make up the essential oil. A fast growing species such as Nicotiana could potentially be grown at relatively high density, allowing a larger amount of plant material to be produced for extraction. After the plants have grown and produced the desired compounds, the plant material could be harvested and the essential oil extracted using an established method such as steam distillation. One advantage of this approach is that the downstream extraction process would be similar to methods already used in the essential oil industry.

An alternative would be to use bacteria or yeast in a fermentation system. In this case, the microorganism could potentially be engineered to produce the desired aromatic compounds through its own metabolic pathways. Nutrients and other required components would be supplied to the fermentation vessel, the microorganism would grow and produce the target compounds, and the resulting compounds would then be recovered from the fermentation system.

One of my main concerns with a microbial system would be whether the essential oil compounds could become toxic to the microorganism. Because many of these compounds can affect cell membranes and other cellular processes, there may be a concentration at which continued production becomes detrimental to the host. One possible solution could be to continuously or periodically remove the product from the fermentation system as it is produced. This could reduce the concentration of the product in contact with the cells and potentially allow production to continue for a longer period.

For an initial project, I would be interested in comparing these approaches and determining whether a plant system or a microbial fermentation system would be more practical for producing a specific essential oil compound.

3. How would you use automation?

Automation could be used with any of the living system options I proposed. For a bacterial or yeast system, liquid handling robots could be used to automate tasks such as preparing growth media, inoculating cultures, transferring samples, and feeding cultures. A bioreactor could also automate environmental conditions such as temperature, pH, oxygen levels, and nutrient availability during fermentation.

Automation could be particularly useful for sampling and measurement during fermentation. For example, samples could be automatically collected at regular intervals and analyzed to determine the concentration of the desired essential oil compound. This information could then be used to automatically adjust the fermentation conditions or determine when product should be removed from the system. In a continuous fermentation system, automation could control the addition of nutrients and removal of culture or product to maintain conditions that support continued production. This could help maintain a productive culture while also preventing the concentration of the essential oil compound from becoming harmful to the microorganism.

Automation could also be applied to the plant system. Automated growing systems could control environmental conditions such as light, temperature, humidity, watering, and nutrient delivery. Cameras or other sensors could monitor plant growth and health, allowing the system to identify when plants are ready for harvesting. Automated harvesting and extraction could then potentially be used to process the plant material and recover the essential oil.

Overall, I would use automation to create a system in which growth, production, sampling, measurement, and product recovery could occur with minimal manual intervention. This would allow many cultures or plants to be monitored simultaneously and would make it easier to collect consistent data and optimize the conditions for essential oil production.

4. How much would you need to make, and how long would it take?

Because essential oils are complex mixtures of many different compounds, I will use limonene, a major component of several essential oils, as a representative target compound rather than attempting to calculate production of an entire essential oil. For this back-of-the-envelope calculation, I will focus on the microbial fermentation approach because there is published production data for limonene using engineered E. coli, which allows me to make a more realistic estimate.

4a. How much?

My initial goal would be to produce 1 kg of purified limonene as a demonstration of a scalable biomanufacturing process. I chose 1 kg because it represents a meaningful amount of product while still being a reasonable target for a hypothetical scale up from laboratory production. A published study using engineered E. coli reported a limonene titer of 3.6 g/L and a space-time yield of 0.15 g/L/hour. (Rolf et al., 2020) Using the reported limonene titer, the approximate fermentation volume required to produce 1 kg of limonene would be:

Target production = 1 kg = 1,000 g limonene

Published titer = 3.6 g limonene/L

1,000 g ÷ 3.6 g/L = 277.8 L

Therefore, I would need approximately 278 L of fermentation capacity to theoretically produce 1 kg of limonene at the published titer. This calculation assumes that the published 3.6 g/L titer could be maintained when scaling the process from the approximately 3 L reactor used in the study to approximately 278 L. This represents roughly a 90-fold increase in fermentation volume, so the actual production volume could be higher if productivity decreases during scale up.

4b. How long?

The same study that was mentioned in the previous question reported a space-time yield of 0.15 g/L/hour. Using the estimated 278 L of fermentation capacity, the expected production rate would be:

278 L × 0.15 g/L/hour = 41.7 g/hour

The amount produced over 24 hours would therefore be:

41.7 g/hour × 24 hours = 1,000.8 g

This is approximately 1 kg of limonene.

The study’s optimized fermentation also produced limonene for approximately 24 hours, reaching its final reported concentration at the end of the fermentation. Therefore, based on these published results, I would estimate approximately 24 hours of active fermentation to produce 1 kg of limonene at the theoretical 278 L scale. (Rolf et al., 2020)

However, the total manufacturing time would be longer than the 24-hour fermentation itself because it would also require time for inoculum preparation, sterilization, fermentation setup, product recovery, purification, and cleaning. In addition, maintaining the same production rate at a roughly 90-fold larger scale is an assumption that would need to be experimentally validated.

For prototype scale development, I would use a desktop liquid handling robot and small fermentation vessels to test different engineered strains and fermentation conditions in parallel. This would allow many conditions to be screened before moving to a larger production system.

For manufacturing at scale, I would use industrial bioreactors with automated control of temperature, pH, oxygen, agitation, and nutrient feeding. I would also use automated sampling and product monitoring, along with in situ product removal. The latter would be particularly useful for limonene because the published process used a two liquid phase system to capture limonene as it was produced. This could help reduce product accumulation in the culture and allow the microorganisms to continue producing limonene.

5. What don’t you know yet?

There are several things I would need to determine experimentally before I could know whether this biomanufacturing system would actually work at a larger scale. The three biggest unknowns for me are the tolerance of the organism to a desired aromatic compound, whether the production rate can be maintained during scale up, and how efficiently the desired aromatic compound can be recovered from the fermentation system.

Source:

Rolf, J., Julsing, M. K., Rosenthal, K., & Lütz, S. (2020). A Gram-Scale Limonene Production Process with Engineered Escherichia coli. Molecules, 25(8), 1881. https://doi.org/10.3390/molecules25081881