Team:NYU Shanghai/Demonstrate

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Magenta Sulfite Colorimetric

Method introduction:

After the bacteria has successfully transformed, we need to test the feasibility and efficiency of our pathway by monitoring the change of methanol concentration of methanol solutions with the transformed E.coli at specific time points. At first we proposed to use gas chromatography. This precise method is commonly implemented in organic chemistry to separate easily vaporized organic compounds and determine their relative amount. However, the gas chromatography machine in our lab cannot test water solutions because the water in the solution will damage the machine due to its polar nature.

Thus, we moved on to an alternative method: Magenta sulfite Colorimetric, which is a commonly used chemical method to detect the methanol concentration in the alcohol industry. The basic mechanism consists of three different solutions: 1) potassium permanganate-phosphoric acid solution which oxidizes methanol into an aldehyde

The second oxalic acid-sulfuric acid solution, removes manganese dioxide produced from oxidation and the un reacted KMnO4.

The third, aniline red- sulfinic acid solution reacts with the produced aldehyde to change the color of the solution, and as a result the absorbance at 590 nm will change (observed with the spectrophotometer).

The first step is to build a standard curve to find the relationship between the absorbance at 590nm and the methanol concentration. Solutions of known concentrations will be treated with the three solutions, and then the absorbance is measured. Then, the transformed bacteria will be added into a methanol solution, after certain time intervals, the three solutions will be added. The E.coli will be removed, and then the solution will be diluted (the method is only accurate for small amounts of methanol, thus the solution must be diluted) and the absorbance will be taken.

Results:

1) standard curve

Methanol amount (mg)	0.06	0.12	0.18	0.24	0.30
Absorbance(A)	0.138	0.363	0.674	1.069	1.245

2) Absorbance at 590nm over time

Time	0min	15min	30min	45min	60min
Absorbance(A)	0.277	0.291	0.262	0.276	0.221

Table 1: The absorbance at 590nm over the period of time with 0.125mg methanol in water solution

Time	0min	15min	30min	45min	60min
Absorbance(A)	0.777	0.904	0.736	0.770	0.835

Table 2: The absorbance at 590nm over the period of time with 0.250mg methanol in water solution

Time	0min	15min	30min	45min	60min
Absorbance(A)	0.843	0.873	0.810	0.819	0.852

Table 3: The absorbance at 590nm over the period of time with 0.250mg methanol in water solution

Time	0min	15min	30min	45min	60min
Absorbance(A)	0.373	0.335	0.575	0.447	0.117

Table 4: The absorbance at 590nm over the period of time with 0.250mg methanol in LB solution

Discussion:

From the standard curve test, we confirmed that the absorbance at 590nm has a linear relationship with the mass of methanol; moreover the absorbance should fall as the methanol amount falls. Therefore, a decreasing absorbance over a period of time proves the validity of our pathway. However, when water was used as our solvent for the methonal, the absorbance fluctuated up and down (Table 1 and 2). This might be because in the dilution process ,the methanol solution was not evenly distributed after centrifugation and only 0.005ml or 0.0025ml out of 1ml was taken out for the testing. So for the following experiments (table 3), instead of directly taking out a small amount of the centrifuged solution, 0.5ml of the upper clear solution was taken out, vortex( ensure equal methanol distribution) and then 0.005ml or 0.0025ml was taken out and diluted for testing. In table 3, a small fluctuation can still be seen, but has significantly decreased compared to table 1 and 2, a falling trend in absorbance can’t be found. According to our survival test result, we found that E.coli survives under 10% methanol water solution for up to an hour, but the colonies are quite small, so we guessed that the bacteria might not function properly under methanol water solution, thus for our last trial, LB was used as the solvent to make the methanol solution instead of water to give a better environment for E.Coli. However, huge fluctuations appeared again, also when the oxalic acid-sulfuric acid solution was added, the reaction was much more vigorous than before, indicating that more of the KMnO4 did not react. After some research, it was found that LB contains an amount of Vitamin B which has carboxyl groups that could interact with the hydroxyl group of methanol, generating an ester bond that can not be oxidized by KMnO4.So this method is not the optimal choice if LB is used as the solvent. Research also shows that gas chromatography is the best method for accurately detecting methanol amounts, however the gas chromatography machine in our lab can only test pure organic solutions. Due to the limitations of our method and time we were not able to test our pathway in higher concentrations.