Team:Paris Bettencourt/Project/Manufacturing

Ferment It Yourself

iGEM Paris-Bettencourt 2O15

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Manufacturing

Background	Aims	Results
Our strains has to be grown in India and to be distributed to the users.	The goal of the manufacturing project is to find a way to grow and distribute our strains cheaply and easily.	We shown that S. cerevisiae mCherry, Lactococcus Lactis and Propionibacterium freudenreichii can grow on rice water and potato water We have design a rice flour cube, that we named VitaCube We have shown that the yeast and the Lactococcus can survive in the VitaCube during at least 4 days We have design a rice flour cube, that we named VitaCube

The aim of our manufacturing project was to find a way to grow and distribute our strains easily and cheaply to reach as many people as possible. The product has to be simple and minimize the disruption of Indian habits.

Strains growth

Our project is designed for poor regions: the cheaper our product, the more people it can reach. Starting from this point, we didn't want to build labs in India, buy media and grow our strains in expensive structures. Therefore, we had to think of a cheap solution to grow the strains in a homemade media, made of common ingredients, cheap and readily available.
To avoid expensive and time-consuming treatment, an edible medium is sought. It could be directly used to cook either our distribution product or the Idli directly. The process would hence be quite simple and accessible to the population.

We worked on 3 different strains: S. cerevisiae mCherry, Lactococcus Lactis and Propionibacterium freudenreichii, which are close from the one we were engineering. After some research, we selected 3 different homemade media to test with our 3 strains:

• Water from the cooking of potato (potatoes cooked in 2L of water for 20min)
• Water from the cooking of potato+sugar(10%)
• Water from the cooking of rice (200ml of rice cooked in 2L of water for 10min)

We also wanted to see if the concentration in potato had an influence so for each media made out of potato water, we tried two different concentrations of potato: 190g/L (named potato dilute) and 250g/L (named potato concentrated).
For each strain, we compared the growth of the strain in the homemade media with the growth in its "normal" media (respectively YPD, M17+Glucose 1% and YEL).
The results can be seen in Figure 1.

As we can see in the first figure, the strains are growing in each media. The rice media is the best one, giving even better result than the normal media for Lactococcus Lactis and Propionibacterium freudenreichii. We couldn't show that neither the concentration of potato nor the addition of sugar had any influence on the growth of our strains.
Eventually, we shown that every strains was growing in potato water and rice water.
Figure 1

Packaging and distribution

One stake of our project is to give the strains to the population. Either community labs or users grow the strains, strains needs to travel from us to the community lab and then to the final user.
To answer this problem, we tried several methods, premilinarily working on a yeast: Saccharomyces cerevisiae mcherry.
We could have just lyophilized the strains but our goal is to design something cheap and easy to do for the locals, using only ingredients they have access to and not time consuming.
We first tried to make different kind of powders, but none of it was satisfying. Powders appeared not to be very convenient.

We realised a powder wasn't the best way to distribute our strains. We found better to make portions, easy to pack, with the possibility to pack several portions together. Portions must be easy to stock.
The most efficient and ergonomic shape appeared to be a cube. Moreover, the cube will mainly be added to Idli, made of rice, so rice flour seemed to be a logical ingredient, consonant with the dish, common in India and cheap.
Little by little, we succeeded to design an easy recipe, to cook small cubes made of rice flour and water: the VitaCubes. The idea was also to be flexible to every VitaCube maker means, therefore the recipe is not very strict and can be adapted to what the people have available.

Now that we have found a convenient distribution mean, we need to be sure that it keeps our strains alive and to know how long it can be stored.
We conducted several survival test on the VitaCubes, using S.cerevisiae mCherry and L. lactis. To make these test, we were plating the liquid solution containing the strains before adding it to the flour, in order to know how many cells we put in the VitaCube. After a define period of drying, we immerse the VitaCube in 10ml of osmosed water and plated the solution obtained to count the numbers of cells that survived in the cube.
At first, we obtained very different results, even if the cubes were all plated after one day of drying. We realised the time of immersion of the cube in the water had an influence over the survival rate: the longer it was, the bigger was the survival rate. This is due to the revival of cells once back in water.
Therefore, we designed a one week experiment: we made plenty of VitaCubes on the same day, and then each day, we plated one cube. Each day, we respected the very same conditions, every step was timed.

Figure 2

Figure 3
On graph 2, we can see that the yeasts are very resistant and have a very high survival rate, decreasing very slowly over time. The survival rate is even higher than 1 because yeasts are growing very fast, so in 20 minutes in the water, they divide. In conclusion, the yeasts can be stored more than one week in a VitaCube.
On graph 3, we can see that L. lactis survival rate is low since the first day and is decreasing very quickly every day. After 4 days of drying, the survival rate started to be very very low. Nevertheless, there are still around 10⁵ cells in a VitaCube after one week, each is far enough to make a culture from it.
Looking at the graphs, we can see that the mix of both strains doesn't influence their survival.

Micro-organisms in idli

We want to make sure that the organisms we are using can grow on idli batter in the conditions of fermentation, and that they can grow together in the same batter. To test this hypothesis, we did two different experiments. The first one consisted in inoculating the different strains of micro-organisms that we intend to use for fermentation in the batter of idli at the moment of fermentation (after the mixing and grinding step). Each time, we add between 10⁷ and 10⁸ cells.

The second experiment consisted in measuring the growth curve using a TECAN imager, to check if the strains that we want to use can grow in such an unusual medium. Here we used classic microbiologic media (LB, MRS, M17, YPD) and unusual DIY media (sterilized osmosed water, sterilized idli water, sterilized idli batter diluted 1000 times).

Growth during fermentation of idli

TECAN study

As we can observe, the E. coli strain we engineered to produce vitamin B2 and the S. cerevisiae who have undergone the PHO 80 or/and PHO85 genes deletion grew well on idli water. This media was obtained by taking the transparent supernatant after decantation of the idli batter.