Autolysis in combination with bead-milling Results

Free amino acid estimation with rFAN assay

Samples from Experiment Cell_extraction_with_autolysis_combined_with_bead-milling.pdf were used for the analysis.

Yeast extract is mostly obtained by autolysis ¹. In autolysis cells digest their own cell compounds with their own enzymes ². The idea was to transfer this commonly used principal on algae. Therefore, C. vulgaris and C. sorokiniana were heated to 50 °C in alkaline or acidic environment for 41 h. To further crack the cell wall, both algae were treated with bead-milling afterwards. To quantify the success of cell wall disruption free amino acids were measured with rFAN-assay.

The yield of free amino acids was set into relation with the amount of biomass used in the experiment (figure 1).

Figure 1 -Autolysis and subsequent bead-milling of algae C. vulgaris and C. sorokiniana. The percentage of free amino acids [%] relates to the biomass used in the experiment.

The highest amounts of free amino acids were reached with yeast at pH 12 with 4.85 %. Both algae showed very low yield in free amino acids. The best results showed C. sorokiniana at pH 12. It is possible, that the amount of glass beads and the size of the glass beads were to little, which led to less cell wall disruption. Therefore, amino acids would have been retained within the cells. This would explain the little amounts of free amino acids achieved with this method. Also, C. vulgaris and C. sorokinia have a cell wall, in contrast to yeast ³>. Cell walls are harder to break, than a plasma membrane. This could explain the difference between the yeast samples and the algae samples. Due to the low yield in free amino acids, it was decided to investigate other methods for cell extraction of algae.

Anthrone assay to Determine Soluble Carbohydrate Concentration

Similar to the rFAN assay the anthrone assay is a method to detect free monosaccharides in a liquid. Therefor samples from the experiment Experiments_AnthroneAssay.pdf were analyzed. Hereby a calibration curve with known amounts of glucose is created (Figure 2, left side). This calibration curve creates the possibility to calculate the sugar concentration of the samples (Figure 2, right side).

Figure 2: Pictures of the anthrone calibration curve as well as the anthrone assay of samples. For the calibration curve known amounts of glucose is dissolved in water and the optical density at 620 nm is measured (left side). This can be used to determine the monosaccharide concentration of anthone treated samples which previously underwent autolysis (pH3 or pH6) with or without subsequent bead-mill treatment (RKM) (right side).

One can tell from the coloring of the samples in figure 2, that the carbohydrate concentration should differ very slightly between the samples pH3, pH6, bead mill extraction +pH3 and bead mill extraction +pH6. Due to the cloudiness of the control sample, a background corrected optical density could not be determined. Therefore, the coloring scheme served as evaluation for successful carbohydrate determination.

Hereby, bead-mill (RKM) with subsequent autolysis at pH3 was determined to be the method of choice.

CDW correlation of algae Chlorella vulgaris Results

By plotting the measured optical densities against the means of the calculated cellular dry weights, a correlation was obtained. It is shown in the following figure.

Figure 1 - OD-CDW correlation of the algae Chlorella vulgaris. Mean of cellular dry weight in g/L (n=2) was plotted against the measured optical density at 750 nm. Trend line was shown in red.

The trend line in figure 1 is poorly matching the trend of the measurement points. For this reason, the correlation curve was rejected. For improvement of this experiment, measurements should be performed only by one experimenter to reduce pipetting errors or other handling mistakes. Also the measurements should be taken over a longer time period to gain more trust worthy results.

CDW-OD correlation by dilution Results

In the following table you can see the calculated cell dry weights with the corresponding optical density of the tubes. Some tubes broke during the experiment so corresponding measurements could not occur.
Table 2 -Calculated values of the cellular dry weight in g/l with the corresponding optical density measured at 750 nm.

OD	CDW[g/l]
4.87	0.98
4.41	0.88
4.44	0.91
4.02	0.8
3.92	0.82
3.52	0.68
3.57	0.71
3.03	0.57
2.65	0.49
2.8	0.5
2.43	0.48
2.56	0.46
2.29	0.4
2.28	0.4

The cellular dry weight in g/l was then plotted against the optical density measured at 750 nm. The plot with the corresponding trend line is shown in the following figure.

Figure 1 - Cellular dry weight in g/l is plotted against the optical density measured at 750 nm. The linear fit is shown in blue together with its formula.

The slope of the formula further been used for fast estimation of the CDW by measuring the optical density at 750 nm.