Project Scale up

We wanted to investigate if the idea of using E.coli to produce fine fragrances is economically viable at an industrial level and to see if it could compete with the current methods available.

In order to do this we first needed to think about what factors and costs would be involved in scaling up the project. We considered the following options;

> Type of growth medium to use to grow the E.coli

> Size of the fermenter needed

> The overall running costs

> How we can purify the product

> Waste products produced

> How the costs of producing fragrances through E.coli compares to fermenting natural crops

Growth Medium:

After looking into various growth mediums that E.coli can grow in, it was thought that the cheapest option would be to use LB (Lysogency Broth). LB Is made from Tryptone, Sodium Chloride, yeast extract and distilled water.

An additional thought was that if the plant was located next to other industry's then some of their waste products could be used in the culture e.g. a brewery, wasted yeast could be used. This would help to cut down costs and help reduce waste for the environment.

Cost analysis:

Zingiberene has been used as the example fragrance in the cost analysis of the scale up. The fragrance company, Givaudan, informed us that red ginger oil made in industry costs £1000 per litre, so we have taken this as a price to compare our product to.

We would need to grow our strain of E.coli in a fermenter, and so it is important to know the size of the fermenter we would need in order to get a good yield of fragrant compounds. In order to do this we used figures from literature and estimated values for the rate that E.coli can produce Zingiberene. We estimated that it can be produced at a rate of 40.8mg/L over 48 hours. When this is divided by zingiberene's density it was shown that we would need a 20,000L tank in order to produce 1L. So effectively we would be producing a product worth a £1000 using a 20,000L tank over 48 hours, we concluded this is not an economically viable option!

However we realised that it isn't a lost cause! To overcome the low yield we need to look at the bigger picture. Our project is in early development stages and we believe the pathway can be further optimised by targeting the rate limiting steps in the pathway and adding the mevalonate pathway from plants into the Ecoli to increase the concentration of important intermediates. Furthermore we can:

> Increase productivity on the enzymes by optimising strength of the promoter

> Attempt to increase activity synthase through site activated mutagenesis

> Optimise growing conditions for the E.coli

> Optimise downstream processing

> Look into methods of downstream processing recovery

If these steps can be taken then it could be possible to increase the concentration by X100 fold. If this was the case then it would be possible to produce the same concentration of fragrant compound in a 200L tank instead. This is a more viable option and could be economically viable.

Product Separation:

In order to extract the fragrance from the E.coli culture we needed to think about which extraction method would be most suitable and economically advantageous. From looking at current fine fragrance production it was drawn to our attention that in order for the product to be considered a fine fragrance, the extraction could not involve any chemicals. This ruled out the most economical option of solvent extraction, so instead we decided steam distillation would be the better method to use. Steam distillation would have to be carried out on small batches of culture from the main fermenter; we would pass steam through the culture causing the volatile fragrant compounds to be driven out of the medium. The product can then be easily separated from the water, and even the waste water collected can still be of value as it contains traces of the fragrant compounds.

Waste Products:

The major waste products from our process will be carbon dioxide and used E.coli culture. We believe that the used E.coli culture however will not be a hazardous waste product as the E.coli will have been killed during the steam distillation process. Therefore we are lead to believe that it can be disposed of into normal waste disposal and water supply. Water companies check and regulate water through the coliform index which rates water purity based on the count of faecal bacteria in the water. If this is however a problem then we would need to take into consideration waste treatment and have a treatment facility on site.

How does using E.coli compare to distillation of plants?

In order to get perspective on the economic viability of the project we compared the costs of using E.coli to the costs of naturally distilling plants to produce fragrant compounds. Looking at lavender fragrance production figures show that suppliers can earn £5000 per acre from 450Kg of dried lavender buds, these buds would then need to be distilled using a purification method. It is estimated that lavender essential oil can be produced at a yield of 0.5% so this would mean that suppliers could make 2.2L of essential lavender oil from one acre of land. However from these costs factors that would need to be included are:

> Farming Labour

> Pesticides and maintenance costs

> Downstream processing costs

> Cost of having a low yield some years due to poor weather conditions

So when comparing natural distillation of plants to synthetically producing the fragrance in E.coli it could be shown that the two methods are not too dissimilar in economic value.

Summary:

Though our analysis above, we recognize that the fragrances that our project target; zingiberene, R-linalool and limonene, using E.coli may not necessarily come at an economical advantage compared to current methods and practices used in Industry. However we do strongly believe that through optimisation of the methods we have used E.coli could be an economically viable option in essential oil production. We think that in the future E.coli could be used to generate fragrances that cannot be easily obtained naturally or chemically synthesised such as the valuable fragrance Ambrox. A synthetic biology approach using E. coli will also be more flexible and will allow consumer and environmental benefits current methods do not offer. We therefore see our project as a proof of concept that E.coli could well be used in the future for essential oil production.

Below is a schematic of the scale up: