Team:Toulouse/Results
Device
Device : physical tests
TPX Bag
Gas diffusion tests
In order to know if our test that we described in “Protocol” test functions, we made a control with acids solutions in a falcon and we sample gas in balance.
For butyric acid we did not detect gas butyric acid by NMR, the solution we used to solubilize gas should be not basic enough. So we made a test with a TPX® bag containing butyric acid into a solution of sodium bicarbonate. As a control we sampled directly a solution of 4% (V/V) of butyric acid.
Figure 1: NMR Spectrum of butyric acid liquid control in red and butyric acid liquid which passed through TPX bag in blue. Red curve is zoomed 1340 times more than blue curve. Each condition was tested in two replicates.
Table 1: Concentrations of butyric acid corresponding to NMR spectrum
Thanks to these results, TPX® overlook butyric acid
outside the bag. We detect only a small quantity but
an optimization of test could be made or a plastic with
bigger porous could be use.
For formic acid we were able to detect it
in gas, probably because its pka is lower than butyric acid.
Figure 2: NMR spectrum of formic acid gas control in red and formic acid gas which passed through TPX bag in blue. At the left top internal standard shows that it is the same scale for both curves. Each condition was tested in two replicates.
Table 2: Concentrations of formic acid corresponding to NMR spectrum
According to these results, TPX® overlook 56 % of formic acid outside the bag in gas phase. We show that formic acid can go through TPX plastic. And with a better test, as we proposed above this percentage could increase.
Device: biological tests
In the end, our objective is to have a bag which contains bacteria to produce alternately butyric acid
and formic acid during at least ten days in order to be practical for beekeeper.
So we faced with some biological questions as:
- Could bacteria live during ten days in micro-aerobic condition?
- Which carbon source could we have to produce continuously acids?
- Would acids be toxic for E.coli?
Characteristics of E.coli growth
In order to know better the E.coli strain we would use for our project, we made a culture in aerobic and micro-aerobic conditions. We sampled OD and supernatant as it is explained here to see what happen in it.
Micro-aerobic condition is obtained thanks to cultivation in specific falcons with holes recovered by a membrane into the plug which let pass oxygen without opening the falcon. They were incubated at 37 °C without agitation to best correspond to our real condition.
Aerobic condition is obtained with classic Erlenmeyer incubated at 37 °C with agitation.
For the medium, we use a minimal medium M9 because we want to follow acids production by NMR. And we choose a standard glucose concentration, 15mM.
Biomass, substrate and products
In order to plot biomass concentration it is necessary to convert the OD measured.
This equation was used:
$$ X=OD_{600nm}\times 0,4325 $$
Where X is the cell concentration (g.L-1)
For substrate and products concentration we plotted peak area of each molecule on NMR spectrum.
Then, we calculated concentration with this equation:
$$[A]=\frac{Area_{molecule}}{Area_TSP} \times [TSP] \times \frac{\textrm{TSP proton number}}{\textrm{A proton number}} \times DF $$
- [A] = concentration of molecule in our solution in mM
- AreaTSP = 1
- [TSP] = 1.075 mM
concentration of Trimethylsilyl propanoic acid in NMR tube, internal reference for quantification - TSP proton number = 9
- DF = Dilution Factor = 1.25
Thanks to these calculations we were able to plot biomass, substrate and products depending on time.
Figure 1: Results of aerobic culture. Culture of BW25113 in M9 medium with [glucose] = 15 mM, in Erlenmeyer at 37°C
Figure 2: Results of micro-aerobic culture. Culture of E. coli BW25113 in M9 medium with [glucose] = 15 mM, in Falcon at 37 °C
Glucose is consumed approximately at the same rate for both conditions but it is not use for the same thing at all. In aerobic condition biomass reaches 3 g/L whereas in micro-aerobic condition there is six times less biomass. Inversely, there are far less products in aerobic conditions, and bacteria consume them when there is not glucose anymore, than in micro-aerobic condition.
For our objective to produce acids in a microporous bag, it is a really interesting results to have naturally bacteria which have slow growth and fermentation products.
We can convert formate
concentration into formic
acid to know how much more
we will have to produce to
kill varroa. Indeed, the bacteria
produce a base but it is the acid that
interests us.
The formula below
is used:
$$ pH=pKa+log \left(\frac{C_{b}}{C_{a}} \right) $$
- pH: medium used is buffered with a low concentration in acid. pH = 7.
- pKa: 3.7 for formic acid and 4.81 for butyric acid
- Cb: base concentration
- Ca: acid concentration
As it is said in the “Eradicate” part, our goal is to produce 50 µM of formic acid to kill varroa, thanks to the equation (3) we know it corresponds to 77,7 mM of formate.
At the maximum the bacteria produces 32mmol/L of formate. It is necessary to add genes involved in formate production to regulate production and multiply it by 2.4. For a perfect regulation it would be necessary to delete pfl-B in E.coli genome not to have formate production during the day.
Bacteria survival
As it is explained here we plated bacteria on Petri dish to know if they were alive or not because OD measure cannot discriminate alive bacteria from dead. This test show us that wild type bacteria can easily survive during at least 15 days. So if we bring them a carbon source during this period they should survive even better.
Figure 3: Bacteria survival results from culture test with BW25113 on M9 with 15mM of glucose during 15 days to mime real survival condition.
Choice of carbon source to produce acids during 10 days
Characteristics of Biosilta kit
En Presso B is a technology which permits to produce a lot of recombinant proteins thanks to a low substrate delivering during 24 hours. This technology is based on polymer degradation by an enzyme which permits to have the right quantity of substrate at each moment. We would like to use this technology to cultivate our cells during one or two weeks in good conditions in order to produce butyrate and formate. The medium with the polymer is solid and contained in separate bags. To know which quantity of butyrate and formate we can produce, we have to know the quantity of substrate we could obtain with the polymer so we made a kinetic test with a high enzyme concentration (50 U/L).
Figure 4: Kinetic test of enzyme which degrades polymer from Biosilta kit. [Enzyme] = 50 U/L in order to have a complete degradation of polymer.
$$ v_{glucose1}=\frac{[glucose]}{time}=\frac{11.1}{3.97}=2.80 g.L^{-1}.h^{-1} , for [E]_{1}=50 U.L^{-1} (4) $$
