2.1 Introduction
3.5.5 In Depth Ethanol
Due to the small sampling window and the potential for a skewed impression to be gained from this limited window, a selection of populations were further investigated over 24 hours with two hourly intervals, during mid-late exponential growth (between 6 and 14 hours after inoculation). The strength of the rate yield changes observed and reviewed in Chapter Two resulted in selection of the populations A8, B5 and C10 for further investigation. SCE2 S. cerevisiae was measured alongside as a Crabtree-positive reference.
To establish ethanol production of each yeast population the ethanol over time was plotted. Replicates, both separate plates and the three samples per plate for the ancestor, were pooled. Then for added statistical robustness the evolved lines data were also pooled and statistical tests carried out of all evolved lines versus the ancestor for each of the four populations subjected to this experimental condition. This allows for the testing of systematic changes in all evolved lines compared to the ancestor. However, the statistical power from this experimental design did not allow for the detection of changes in single lines.
59
Figure 3.2 and Table 3.3 present the ethanol concentration changes demonstrated over a 24-hour period. Yeast population A8 demonstrates a significant decrease in ethanol concentration overall from hour 8 through to hour 24. This is surprising as the previous experiment suggested that there was an increase in most lines. It is possible that the higher ethanol concentrations in experiment 1 are observed because faster growth in the evolved lines means these lines were already at a higher population size.
Yeast population B5 demonstrates only one significant change over the 24 hours. At hour 6 there is a significant increase in ethanol concentration in comparison to the ancestor. While the trend appears to continue towards increasing, no significant change from the ancestor is exhibited.
Figure 3.2: Average Change in Ethanol Production (g/L) per Population. Comparing evolved lines with the ancestor. Over 24 hours each of the four yeasts was sampled along with 3 repeats of the ancestor across two repeat plates. Each yeast is represented by a specific colour as shown in the key. Significance in marked with an asterisk.
60
With yeast population C10 there is no apparent change in ethanol concentration over the entire 24-hour period. Lastly, SCE2 demonstrates a different trend over the 24-hour sample period. Initially at hour 4 through to hour 6, there is a significant increase in ethanol concentration
Species Sample Time (hrs) D-value T-value P-value
0 0.000 -0.908 0.397 6 0.000 -0.898 0.424 8 -0.002 1.612 0.159 A8 10 -0.001 0.629 0.553 12 -0.006 2.948 0.028 14 -0.017 2.943 0.022 24 -0.001 4.427 0.004 0 0.000 -0.814 0.446 6 0.000 1.087 0.315 8 -0.001 1.446 0.193 B5 10 0.003 -3.545 0.010 12 0.005 -1.611 0.167 14 0.005 -1.659 0.150 24 0.000 -0.356 0.735 0 0.001 -1.311 0.235 6 0.000 1.508 0.177 8 0.000 -0.319 0.772 C10 10 -0.001 0.466 0.660 12 0.001 -0.217 0.836 14 -0.004 0.907 0.395 24 0.000 1.421 0.198 0 0.000 -0.615 0.558 6 0.008 -2.239 0.062 8 0.018 -2.751 0.031 SC 10 0.029 -2.948 0.035 12 -0.001 0.292 0.779 14 -0.017 2.269 0.066 24 -0.008 2.555 0.040
Table 3.3 Statistical Significance of Changes of Ethanol Concentration over 24 hours. Unpaired, unequal variance t-tests run on all the evolved lines versus the ancestor of four of the yeast populations. Statistically significant results are highlighted in bold.
61
compared to the ancestor. After this, the concentration decreases compared to the ancestor to result in a significant decrease at hour 24. This suggests that rather than a change in ethanol production the change is in the shift in timing of the peak ethanol point, i.e. the growth time has shifted to be faster than the ancestor and therefore reaches the peak ethanol earlier in the experiment than the ancestor. This is somewhat surprising because the growth data from Chapter Two do not suggest the evolved lines grow faster.
3
3.5.6
Summary
The experiments discussed above aimed to investigate the ethanol concentration change between the evolved and ancestor lines. Two experiments were carried out, firstly ethanol was monitored through the entire course of the experimental evolution to track any changes in real- time and then analysed back to back to eliminate plate variance. Secondly four populations were selected for more intensive study over a longer period of experimental monitoring. From the first experiment, no clear picture could be determined due to a small window of time being sampled. Therefore, A8 Kodamaea, B5 Pichia kluyveri, C10 Issatchenkia and SCE2 S. cerevisiae were further tested with more intense sampling carried out. The lack of statistical power in these experiments means that a conclusion cannot be made on the differences between ancestors and individual lines. In terms of systematic changes that affected all evolved lines of a given ancestor, only B5 seems to show a small increase in ethanol production, while A8 appears to decrease. From this it is concluded that without further investigation no notable results were observed. To achieve this, stronger investigation into optical density and glucose concentration may help in gaining further insight into the changes in ethanol across the evolution experiment and to clarify if the results here are in fact real observed change. Additionally, it would be of interest to look into other metabolites that could potentially have been excreted into the medium.
Protein Assay
Spectrophotometry is an objective way of measuring absorbance of light of a sample as an expression of wavelength. In a spectrophotometer, the light source is directed into the sample
62
and, upon hitting the sample, is absorbed and the transmitted fraction is measured as the optical density. Using optical density to measure highly concentrated samples is difficult as the observations become non-linear with increasing solution concentration. Moreover, when tracking populations of cells, the absorption behaviour of evolving populations could in principle change, for example, because of differences in cell composition, size and shape. Due to the indirect nature of optical density as measurements for growth yield, it is of interest to validate OD-based results with alternative methods. One alternative is to measure the dry mass of samples taken during growth. However, this technique requires larger samples, and is labour intensive. Optical density measurements can also be validated using colony forming unit (CFUs) counting, a method that will be tested further in Chapter Four. Another alternative is to measure the protein concentration of a culture. Therefore, the method opted for was a colorimetric assay, the Thermo Scientific™ Pierce™ 660nm Protein Assay.