1.1 Furfuryl alcohol effect on glucose fermentation and microscopy

To assess the number of inactive yeast cells "blue staining" is usually done. Methylene blue is the most commonly used color agent. It is a redox indicator that turns colorless in the presence of the active enzymes produced by the yeast Saccharomyces cerevisiae. For verification of the results this compound is compared with erioglaucine (E133) (Brilliant blue No. 1).

In order to verify that erioglaucine is equal in quality to methylene blue, a dose response curve for the toxic compound fufuryl alcohol is produced, and the mortality is then examined with the two color agents. 6 fermentations of 100 ml are started with different levels of furfuryl alcohol (0.0, 1.5, 3.5, 6.0, 9.0 and 15.0) (mL/L) and 2 g/L Turbo yeast (from AlcoTec). All the fermentations are completed in blue cap flasks in Millipore water and with 100 g/L glucose monohydrate. The yeast is left to ferment at 25, 32, 40 and 45°C, using 100 rpm of stirring for 24 hours. Weight loss is measured during working hours to monitor the fermentation. After 24 hours samples are taken for HPLC analysis as described by Thomsen et al. (2009). A methylene blue and an erioglaucine solution are produced each of 0.29 g/L. The 6 samples are divided into 12 portions to perform double repetition. Six portions were mixed with methylene blue in a 9:1 ratio and six portions with erioglaucine in a 1:1 ratio. The samples are examined with microscopy at 1000x magnification just after the color agent was added.