Team:Alberta/EsterificationExtraction

We are proposing to accumulate free fatty acids in Neurospora Crassa in order to produce a usable fuel. Our plan is to convert these fatty acids into biodiesel by esterifying them with methanol and using an acid catalyst. The reaction proceeds in the following manner:

Our procedure uses methanolic-HCL directly on the cell mass in order to esterify the fatty acids in the fungus. A by-product of the reaction is water so we make sure to use dry samples to push the reaction more towards completion. The direct esterification method was chosen because it is a simple method and has been shown to be more efficient an extraction/esterification method then lipid extraction and then esterification of the free lipid, (2, 3). We separated the newly created methyl esters from the reaction by adding water and hexane, the biodiesel will go into the top hexane layer.

Base catalyzed esterification is another method of esterification and generally requires a lower reaction temperature and less reaction time, however this was not a good option for our project because base catalysis does not esterifiy free fatty acids, the very lipid we are accumulating (3).

Another part of our project went into designing a theoretical plant for large scale production of biodiesel. The following is a schematic of what the plant would look like

The design is intended to recycle as much reagent as possible, mainly by removing hexane through negative pressure, and distilling off methanol. The methanol distillation is also important so that that toxin is removed from the water before it is returned back to the water supply.

Overall this project looks at what is necessary to produce a renewable fuel source on a low cost food-stock .

Results:

N.Crassa was grown on three different substrates; Potato-glucose medium, solid paper mill sludge with glucose and fertilizer, and wheat straw with glucose. The purpose was to look for differences in fatty acid metabolism based on these substrates. Cultures were grown for five days and samples were used for Gas Chromatography (GC) analysis using both mass spectrometry (MS) to determine which fatty acids are present, and Flame Ionization detector (FID) GC to quantify the fatty acids. The MS-GC data indicated that all three samples produced predominantly C17, and C19 methyl esters. From the FID-GC data the area under each peak was compared to the area under the standard, whose concentration is known. The following equation was used,

Concentration of Peak=Area under PeakArea under internal Standard Peak×Concentration of Internal Standard

From that the concentration of each peak was calculated, the total amount of fatty acid was determined by adding the concentrations together and multiplying by the volume of hexane used. The Fatty acid flight times between the two GC readings differ, so they have to be matched using their relative position to the internal standard, so it is never certain what peak is actually the fatty acid you are looking for but there are some that are closer than others. This is way in the following table multiple peaks could be either C17 methyl esters, or C19 methyl esters. The figure below shows the raw GC data for both MS (Left) and FID (right) of each sample, the table gives the concentration of each peak on the FID-GC plot, and possible identities based on the MS-GC data. It also shows the total amount of fuel produced per gram of wet weight cell mass.

Discussion:

From the MS-GC all three samples produced C17 and C19 methyl esters, showing that N.Crassa naturally has C16 and C18 fatty acids as the predominate species. From the FID-GC the samples grown on solid sludge, and wheat straw produced more amounts of fuel (per g of wet weight) then when grown on N.Crassa’s preferred Potato medium. The reason for this increase is unknown; however it could be caused by a survival signal in the in N.Crassa when placed in an environment with less accessible food sources then the potato medium, or the fungus grows slower in the cellulosic media so at five days it is still accumulating fatty acid whereas the cells in the potato media have started to break down the fatty acids. In any case we can infer from this data that growth on cellulosic material does not inhibit fatty acid synthesis.

Applications/ Future directions:

In the future this data should be compared against the altered N.Crassa to see if there is an increase in fatty acid levels and/or a shift in those levels due to the addition of a thioesterase, and the knockout of β-oxidation. It would be interesting to determine whether the expression of a shorter chain length thioesterase would produce a shift in the relative amounts of fatty acid.

References:

1. Christie, W.W., PREPARATION OF ESTER DERIVATIVES OF FATTY ACIDS FOR CHROMATOGRAPHIC ANALYSIS, The Scottish Crop Research Institute, Invergowrie, Dundee, Scotland, pp. 69-111 [1993] [Ed. W.W. Christie, Oily Press, Dundee].
2. Lewis, T., Nichols, P.D., McMeekin, T.A., Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs, Journal of Microbiological Methods 43 (2000) pp. 107–116.
3. Griffiths, M.J., van Hille, R.P., Harrison, S.T.L., Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae, Lipids (2010) volume 45, pp. 1053–1060.