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Revision as of 23:52, 24 September 2012

With Gas Chromatography-Mass Spectrometry one can separate and identify different volatiles present in meat that is starting to spoil. We thought that the identification of these volatiles by GC-MS would point out exactly what compounds influence the behavior of our identified promoters [Link to identification page], but we were surprised by the outcome…

The University of Groningen has a lot of GC-MS equipment available and a large commercial database with compounds that we could use to identify the substrates we found in the GC-MS data. So far so good. However, one of the drawbacks of the GC-MS is that the compounds that we might identify from meat that starts to spoil, will be destroyed during the measurements. No further analysis of these compounds is possible then. But if the GC-MS measurements succeed, reliable qualitative data is obtained. These data are hard to analyze due to the large diversity of the volatiles present.



Picture: Arjan and Tom at work with the GC-MS.



Sample bottles with rotten minced meat



Picture: GC-MS setup: All our samples, ready to be measured. The volatiles will be taken up by the syringe in the left corner (red, to the left)

The first step of separation in the GC is based on the fact that different compounds have different boiling points. Therefore, complete evaporation of the compounds should happen all at once and at a high temperature. We used the “headspace method” to search for compounds from meat that was left to rot in a bottle (see picture): after incubation at a high temperature the vapors were extracted from the bottle and injected into the GC-MS. Note: the rotting process of the meat was done in the exact similar way as the microarray experiment was performed for the identification of pBAD-meat promoters [Link to identification page].

In the GC oven, the vapors containing the compounds will condensate, making separation possible. The columns used for GC were HP-5 and HP-1, these are good columns for general use, it has to be noted that these columns cannot identify amino compounds.

HP-5:
dimensions: 30 m x 0,25 mm x 0,25 um
manufactory: Agilent
Column number: 19091J-433
stationary phase: (5%-Phenyl)-95%methylpolysiloxane

HP-1:
dimensions: 30 m x 0,25 mm x 0,25 um
manufactory: Agilent
Column number: 19091Z-433
stationary phase: 100% polysiloxane


After this separation method based on boiling point, the measurement continues with Mass Spectrometry. This is technique enables you to identify compounds based on their differences in mass to charge ratio. Our machine uses electron impact ionization in a vacuum with quadruple separation. This means that the compounds in the machine will be bombarded with electrons in a vacuum. Because of this, they will receive a charge and are most likely to break into pieces. These flying bits and pieces of compounds, with each their own charges, will be prevented from crashing into the wall by the quadruple poles. One can adjust the changing charge of the poles and thereby choose to range of spectrum of the identifiable compounds with their different mass to charge ratio’s.

The reliability of this measurement depends on the range of the spectrum, the reaction(s) with other molecules, which might cause redundant mass charge ratio’s, and how small the differences in molecule structure are, like isomers, that are difficult to separate.

The first GC-MS experiments with rotten meat resulted in blank spectra’s. Unfortunately, the concentration of volatiles was probably too low in the extracted sample for the equipment to measure. However, during the microarray we saw that the bacteria were already able to detect small amounts of volatiles. In the machine, it might have happened that some volatiles were not able to escape from the meat. To obtain more volatiles from the meat we used brine as solvent. It can extract the volatiles, but due to the high salt concentration, it does not allow the volatiles to stay in solution with a higher temperature. Hence, during the incubation most volatiles will evaporate and ?????
But also this did not work out, because we got blank spectra’s again. We soon came to the conclusion that bacteria seem to be able to sense volatiles better than a state-of-the-art equipment like a GC-MS, cannot detect. A very impressive thought! But we did not gave up, though.

The first measurements with only volatiles gave no reliable results. Therefore we decided to dissolve the rotten meat into organic solvents to ensure that more compounds are available for measurement. Furthermore, we used liquid injection instead of the headspace method. Our meat samples were left to rot for more than a day at room temperature, before adding the organic solvent. After addition of the solvent, the meat was left to incubate in the solvent for several hours, while frequently vortexing. The solvent was extracted and filtered before injection in to the GC. But we did not use only one solvent: we needed to study this thoroughly and to cover all the polar and non-polar volatiles, we used different organic solvents.

For an apolar solvent we used toluene and hexane. Toluene gave an emulsion after it was extracted from the meat, in order to prevent damage to the columns we decided to only use hexane as the apolar solvent. Dichloromethane was used as the mid-polar solvent, and methanol as the polar solvent.

Finally, by using this method, we did get interesting results. From both the HP-1 and HP-5 column we got spectra. After the library search only compounds with a quality over 80% are considered reliable. We took the compounds found in the spectra of rotten meat and subtracted the compounds found in the spectra’s of fresh meat and the blank background. A blank measurement is very important in order to distinguish the rotten volatiles from volatiles already present in fresh meat. But also measurements of the solvent only and with fresh meat ensures that background noise was avoided. This approach resulted in the following compounds, origination only from the rotten meat, with a quality that matches with approximately with 80% to the reference library.





We discussed our results with prof. dr. ir. Minnaard, an organic chemist. He mentioned that tridecanoic acid was an interesting find because this is a C30 fatty acid, which is expected not to come of the columns easily. This substrate is also found on both columns and with different solvents.

He also noted that the overall reference quality was low, because results with less than 80% quality are considered as unreliable This is probably caused by compounds that lie close together in the same region of the MS-spectra, making it harder to match them to compounds in the library and thus resulting in a lower quality match.

Some of the compounds found contain a Fluor group, which is rarely found in nature. It’s most likely that these library matches are not correct, thus we excluded these compounds. Also nitro compounds are not likely to occur in these conditions, so these substrate are also not likely to be correct. An explanation for this, lies in the library search. It wants to fit spectra to known spectra in the database, but since our spectra show very uncommon things, our spectra are fitted to known results with the best fit. Due to this overlap, a good fit is not guaranteed and the computer decides ‘blindly’ what the best fitting substrate spectra is. These fits can be incorrect, so we should take critical when we extract conclusions from this results.

Besides the tridecanoic acid, there were several more interesting compounds. Benzenecarboxylic acid was found with multiple solvents and on both columns. Other interesting compounds noted by Prof. Minnaard were Bicyclo[4.3.1]decan-10-one, 1-Hexadecanol and beta.-Phenylpropiophenone.

Ideally, we would like to verify that these compounds are correct. The normal procedure is to acquire the pure substrate and inject into the GC-MS. The spectra’s are compared and a conclusion on the reliability of the data is made. However, due to shortage of time and funds, we chose not do this. Hopefully we are able to peform more experiments in the future, but for now the GC-MS is a interesting part of our iGEM project, but not the major part. We decided to spend our on other research, although prof. Minnaard suggested that we should do another microarray experiment, using these compounds instead of the rotten meat, in the future.

If time allows it, we will try to setup an experiment where we can use the exact compounds to trigger the promoter in the construct and activate the pigment. Instead of using rotted meat during the growth experiment we can inoculate the media with these compounds or pump the fumes into the culture during the growth of B. subtilis with our construct.

We were very lucky that we could borrow these tools during the summer holiday. Therefore, we would like to thank Monique Smith from Bio Organic Chemistry for her valuable help during the measurements and her explanations about the technique.

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