Team:Alberta/EsterificationExtraction

From 2011.igem.org

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     <h3>Outline:</h3>
     <h3>Outline:</h3>
-
     <p>We are proposing to accumulate free fatty acids in Neurospora
+
     <p>We are proposing to accumulate free fatty acids in <i>Neurospora
-
    Crassa in order to produce a usable fuel. Our plan is to
+
        crassa</i> in order to produce a usable fuel. Our plan is to
     convert these fatty acids into biodiesel by esterifying them
     convert these fatty acids into biodiesel by esterifying them
     with methanol and using an acid catalyst. The reaction proceeds
     with methanol and using an acid catalyst. The reaction proceeds
     in the following manner:</p>
     in the following manner:</p>
 +
    <center>
     <img src="https://static.igem.org/mediawiki/2011/8/8c/Alberta-Esterification.png">
     <img src="https://static.igem.org/mediawiki/2011/8/8c/Alberta-Esterification.png">
 +
    </center>
     <p>Our procedure uses methanolic-HCL directly on the cell
     <p>Our procedure uses methanolic-HCL directly on the cell
Line 35: Line 37:
     a simple method and has been shown to be more efficient an
     a simple method and has been shown to be more efficient an
     extraction/esterification method then lipid extraction and
     extraction/esterification method then lipid extraction and
-
     then esterification of the free lipid, (2, 3). We separated the
+
     then esterification of the free lipid<sup>2,3</sup>. We separated the
     newly created methyl esters from the reaction by adding water
     newly created methyl esters from the reaction by adding water
     and hexane, the biodiesel will go into the top hexane layer.</p>
     and hexane, the biodiesel will go into the top hexane layer.</p>
Line 43: Line 45:
     temperature and less reaction time, however this was not a good
     temperature and less reaction time, however this was not a good
     option for our project because base catalysis does not esterifiy
     option for our project because base catalysis does not esterifiy
-
     free fatty acids, the very lipid we are accumulating (3). </p>
+
     free fatty acids, the very lipid we are accumulating<sup>3</sup>. </p>
     <br>
     <br>
     <p>Another part of our project went into designing a theoretical
     <p>Another part of our project went into designing a theoretical
Line 58: Line 60:
     a renewable fuel source on a low cost food-stock.<p>
     a renewable fuel source on a low cost food-stock.<p>
 +
    <br>
     <h3>Results:</h3>
     <h3>Results:</h3>
-
<p>N.Crassa was grown on three different substrates; Potato-glucose
+
    <p><i>N. crassa</i> was grown on three different substrates; Potato-glucose
medium, solid paper mill sludge with glucose and fertilizer, and
medium, solid paper mill sludge with glucose and fertilizer, and
wheat straw with glucose. The purpose was to look for differences
wheat straw with glucose. The purpose was to look for differences
Line 74: Line 77:
<br>
<br>
-
<p>Concentration of Peak=Area under PeakArea under internal Standard
+
<center>
-
Peak×Concentration of Internal Standard</p>
+
<img src="https://static.igem.org/mediawiki/2011/a/ad/GC-equation.gif">
 +
</center>
<br>
<br>
Line 93: Line 97:
amount of fuel produced per gram of wet weight cell mass.      </p>
amount of fuel produced per gram of wet weight cell mass.      </p>
-
<!--<img src="https://static.igem.org/mediawiki/2011/b/b4/Gc-data2.png">-->
+
<br>
 +
<h4>Sludge</h4>
 +
 +
<center>
 +
<table class=figure>
 +
    <tr class=top>
 +
        <th></th>
 +
        <th>Peak Area</th>
 +
        <th>Concentration (mg/mL)</th>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>Standard</th>
 +
        <td>42643197</td>
 +
        <td>0.5000</td>
 +
    </tr>
 +
    <tr>
 +
        <th>
 +
            C17 methyl ester
 +
        </th>
 +
        <td>
 +
            1283841
 +
        </td>
 +
        <td>
 +
            0.0151
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>379375
 +
        </td>
 +
        <td>0.0044
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>428266
 +
        </td>
 +
        <td>0.0050
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>367994
 +
        </td>
 +
        <td>0.0043
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>338038
 +
        </td>
 +
        <td>0.0040
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>C19 methyl ester
 +
        </th>
 +
        <td>1871125
 +
        </td>
 +
        <td>0.0219
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>C19 methyl ester
 +
        </th>
 +
        <td>2963938
 +
        </td>
 +
        <td>0.0348
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>C19 methyl ester
 +
        </th>
 +
        <td>5467430
 +
        </td>
 +
        <td>0.0641
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>791251
 +
        </td>
 +
        <td>0.0093
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>664445
 +
        </td>
 +
        <td>0.0078
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>671449
 +
        </td>
 +
        <td>0.0079
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>1018669
 +
        </td>
 +
        <td>0.0119
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>928668
 +
        </td>
 +
        <td>0.0109
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>318504
 +
        </td>
 +
        <td>0.0037
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>1311464
 +
        </td>
 +
        <td>0.0154
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>332889
 +
        </td>
 +
        <td>0.0039
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>?
 +
        </th>
 +
        <td>452418
 +
        </td>
 +
        <td>0.0053
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>?
 +
        </th>
 +
        <td>399054
 +
        </td>
 +
        <td>0.0047
 +
        </td>
 +
    </tr>
 +
    <tr class=top_bottom>
 +
        <th>Total
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>0.2344
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>mg of FAMES
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>0.4687
 +
        </td>
 +
    </tr>
 +
    <tr>
 +
        <th>mg fuel/g wet <i>N. crassa</i>
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>1.56
 +
        </td>
 +
    </tr>
 +
</table>
 +
</center>
 +
 +
<br>
 +
<h4>Potato</h4>
 +
 +
<center>
 +
<table class=figure>
 +
    <tr class=top>
 +
        <th></th>
 +
        <th>Peak Area</th>
 +
        <th>Concentration (mg/mL)</th>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>Standard</th>
 +
        <td>44476466 </td>
 +
        <td>0.5000</td>
 +
    </tr>
 +
    <tr>
 +
        <th>C17 methyl ester</th>
 +
        <td>752173</td>
 +
        <td>0.0085</td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>C19 methyl ester</th>
 +
        <td>1121567</td>
 +
        <td>0.0126</td>
 +
    </tr>
 +
    <tr>
 +
        <th>C19 methyl ester</th>
 +
        <td>1001098</td>
 +
        <td>0.0113</td>
 +
    </tr>
 +
    <tr class="odd top_bottom">
 +
        <th>Total</th>
 +
        <td></td>
 +
        <td>0.0323</td>
 +
    </tr>
 +
    <tr>
 +
        <th>mg of FAMES
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>0.0646
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>mg fuel/g wet <i>N. crassa</i>
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>0.215
 +
        </td>
 +
    </tr>
 +
</table>
 +
</center>
 +
 +
<br>
 +
<h4>Wheat Straw</h4>
 +
 +
<center>
 +
<table class=figure>
 +
    <tr class=top>
 +
        <th></th>
 +
        <th>Peak Area</th>
 +
        <th>Concentration (mg/mL)</th>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>Standard</th>
 +
        <td>49754071</td>
 +
        <td>0.5000</td>
 +
    </tr>
 +
    <tr>
 +
        <th>C17 methyl ester</th>
 +
        <td>92905</td>
 +
        <td>0.0009</td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>C17 methyl ester</th>
 +
        <td>5222154</td>
 +
        <td>0.0525</td>
 +
    </tr>
 +
    <tr>
 +
        <th>C19 methyl ester</th>
 +
        <td>5953318 </td>
 +
        <td>0.0598</td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>C19 methyl ester</th>
 +
        <td>5973655 </td>
 +
        <td>0.0600</td>
 +
    </tr>
 +
    <tr>
 +
        <th>C19 methyl ester</th>
 +
        <td>1210395 </td>
 +
        <td>0.0122</td>
 +
    </tr>
 +
    <tr class="odd top_bottom">
 +
        <th>Total</th>
 +
        <td></td>
 +
        <td>0.1854</td>
 +
    </tr>
 +
    <tr>
 +
        <th>mg of FAMES
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>0.3709
 +
        </td>
 +
    </tr>
 +
    <tr class=odd>
 +
        <th>mg fuel/g wet <i>N. crassa</i>
 +
        </th>
 +
        <td>
 +
        </td>
 +
        <td>1.85
 +
        </td>
 +
    </tr>
 +
</table>
 +
</center>
 +
 +
<br>
<h3>Discussion:</h3>
<h3>Discussion:</h3>
<p>From the MS-GC all three samples produced C17 and C19 methyl
<p>From the MS-GC all three samples produced C17 and C19 methyl
-
esters, showing that N.Crassa naturally has C16 and C18 fatty acids
+
esters, showing that <i>N. crassa</i> naturally has C16 and C18 fatty acids
as the predominate species. From the FID-GC the samples grown on
as the predominate species. From the FID-GC the samples grown on
solid sludge, and wheat straw produced more amounts of fuel (per
solid sludge, and wheat straw produced more amounts of fuel (per
-
g of wet weight) then when grown on N.Crassa’s preferred Potato
+
g of wet weight) then when grown on <i>N. crassa's</i> preferred potato dextrose
medium. The reason for this increase is unknown; however it could
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
+
be caused by a survival signal in the in <i>N. crassa</i> when placed in
-
an environment with less accessible food sources then the potato
+
an environment with less accessible food sources then the potato dextrose
medium, or the fungus grows slower in the cellulosic media so at
medium, or the fungus grows slower in the cellulosic media so at
five days it is still accumulating fatty acid whereas the cells in
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
+
the potato dextrose media have started to break down the fatty acids. In any
case we can infer from this data that growth on cellulosic material
case we can infer from this data that growth on cellulosic material
does not inhibit fatty acid synthesis.  </p>
does not inhibit fatty acid synthesis.  </p>
 +
<br>
<h3>Applications/ Future directions:</h3>
<h3>Applications/ Future directions:</h3>
<p>In the future this data should be compared against the altered
<p>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
+
<i>N. crassa</i> 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
a shift in those levels due to the addition of a thioesterase, and
the knockout of β-oxidation. It would be interesting to determine
the knockout of β-oxidation. It would be interesting to determine
Line 120: Line 432:
produce a shift in the relative amounts of fatty acid.  </p>
produce a shift in the relative amounts of fatty acid.  </p>
 +
<br>
<h3>References:</h3>
<h3>References:</h3>
<ol>
<ol>
Line 129: Line 442:
     of extraction methods for recovery of fatty acids from
     of extraction methods for recovery of fatty acids from
     lipid-producing microheterotrophs, Journal of Microbiological
     lipid-producing microheterotrophs, Journal of Microbiological
-
     Methods 43 (2000) pp. 107–116.</li>
+
     Methods 43 (2000) pp. 107-116.</li>
     <li>Griffiths, M.J., van Hille, R.P., Harrison, S.T.L., Selection
     <li>Griffiths, M.J., van Hille, R.P., Harrison, S.T.L., Selection
     of Direct Transesterification as the Preferred Method for Assay
     of Direct Transesterification as the Preferred Method for Assay
     of Fatty Acid Content of Microalgae, Lipids (2010) volume 45,
     of Fatty Acid Content of Microalgae, Lipids (2010) volume 45,
-
     pp. 1053–1060.</li>
+
     pp. 1053-1060.</li>
</ol>
</ol>

Latest revision as of 00:07, 29 September 2011

PROJECT

Esterification and Extraction

Outline:

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 lipid2,3. 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 accumulating3.


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,



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. This figure 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.


Sludge

Peak Area Concentration (mg/mL)
Standard 42643197 0.5000
C17 methyl ester 1283841 0.0151
? 379375 0.0044
? 428266 0.0050
? 367994 0.0043
? 338038 0.0040
C19 methyl ester 1871125 0.0219
C19 methyl ester 2963938 0.0348
C19 methyl ester 5467430 0.0641
? 791251 0.0093
? 664445 0.0078
? 671449 0.0079
? 1018669 0.0119
? 928668 0.0109
? 318504 0.0037
? 1311464 0.0154
? 332889 0.0039
? 452418 0.0053
? 399054 0.0047
Total 0.2344
mg of FAMES 0.4687
mg fuel/g wet N. crassa 1.56

Potato

Peak Area Concentration (mg/mL)
Standard 44476466 0.5000
C17 methyl ester 752173 0.0085
C19 methyl ester 1121567 0.0126
C19 methyl ester 1001098 0.0113
Total 0.0323
mg of FAMES 0.0646
mg fuel/g wet N. crassa 0.215

Wheat Straw

Peak Area Concentration (mg/mL)
Standard 49754071 0.5000
C17 methyl ester 92905 0.0009
C17 methyl ester 5222154 0.0525
C19 methyl ester 5953318 0.0598
C19 methyl ester 5973655 0.0600
C19 methyl ester 1210395 0.0122
Total 0.1854
mg of FAMES 0.3709
mg fuel/g wet N. crassa 1.85

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 dextrose 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 dextrose 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 dextrose 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.
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