Team:Alberta/Methodology/Notebook/Esterification

• Overview
• Parts
• Protocols
  • Growth
  • Genetics
  • Extraction and Esterification
• Notebook
  • Growth
  • Genetics
  • Esterification
• Safety
  • Section I.
  • Section II.
  • Section III.
  • Section IV.
  • References

Notebook

Esterification

Weeks 1-3 May 8-29

• Conducted research into methods used to extract and esterify lipids in a cell. Decided on a direct esterification method, using methanolic HCl. Ordered chemicals required and obtained required glass ware.

Week 4 May 30- June 5

• Tested Esterification procedure for around 1g of dry N.Crassa using distilling apparatus, distillation of hexane proved difficult could not isolate large amount of fatty acid methyl esters. Retried experiment with a reflux apparatus, tried to use two samples, 0.75M Methanolic HCl sample, and a negative control of just methanol. Took samples of the hexane layer for GC analysis.

Week 5 June 6-12

• Used the same procedure as above but samples were palm oil and olive oil, used to determine the efficiency of the procedure.

Week 6 June 13-19

• Waited on GC samples, found that samples were not good enough received new protocol from Dr. Stewarts lab, this time using 3M methanolic HCl and smaller sample sizes. Grew cultures of N. Crassa for three days for samples to esterify.

Week 7 June 20-26

• Used the new protocol for GC analysis, set up four samples, two using N. Crassa and methanolic HCL, one using N.Crassa with methanol (as a negative control), and one to esterify palm oil (as a positive control).

Week 8 June 27-July 3

• Previously grew N. Crassa for five days in four different conditions; (1) solid paper mill sludge with glucose and fertilizer, (2) standard potato and glucose medium, (3) grass with glucose and fertilizer, and (4) wheat straw and glucose. Took portions by weight and preformed GC analysis on them using the protocol so far.

Week 9 July 4-10

• Received data from the first GC samples from the new protocol, the palm oil sample was not run on GC columns because of strange colour. The other samples showed strange and inconsistent peaks between mass spectrometry data and quantification data by FID. The suggestion from Dr. Stewart’s lab was to reduce the heating time to one hour, and only extract with one portion of hexane as two does not add much more lipids. Tried this new procedure on the following five day samples (preserved by freezing); (1) solid paper mill sludge with glucose and fertilizer, (2) standard potato and glucose medium, (3) grass with glucose and fertilizer, and (4) wheat straw and glucose.

Weeks 10-13 July 11- August 7

• Designed large scale reactor model, and helped the genetics team.

Week 14 August 8-14

• Tried to esterify a large amount of wild type N.Crassa, started with ~8g of cell mass, ended with 0.075g of biodiesel (~1%). Used only a 1 hour incubation time with one volume of hexane for isolation, the rest of the procedure was the same as the large esterification procedure.

Week 15 August 15-21

• Tried making large amount of fuel again, this time using ~6g of N.Crassa and trying to use less hexane. Ended up with 0.017g (~0.3%), decided to try using more hexane in two batches, and crush the cells beforehand.

Week 16 August 22-28

• Esterified ~23g of N.Crassa using the large esterification procedure, ended up with 0.264g of fuel (~1%).

Week 17 August 29-Sept4

• Esterified more large amounts of N.Crassa to produce a total amount of fuel of 1.394g did this using two more batches, one with ~37g of cell mass and one with ~19g.

Post Sept 4

• Analyzed data from GC experiments.