Chapter 4. Enhancement of butanol tolerance of the selected strain via
4.3 Results and discussion
performed in one adaptation phase till the specific growth rate of the stressed cells matched that of control (unstressed cells). Once this condition was fulfilled, the stress concentration was increased by exposing the cells to higher concentration of butanol, thereby initiating second phase of adaptation, and similar adaptation cycles were carried out.
4.2.8 Analytical methods
The collected sample was centrifuged at 10,000×gfor 10 min at 4°C. The pellet was used to measure growth using UV-visible spectrophotometer (Cary 50, Varian, Australia) as detailed in section 3.2.7. The substrates, organic acids, and solvents concentrations present in the supernatant were measured in high performance liquid chromatograph (HPLC, LC-20AD, Shimadzu, Japan) equipped with a Rezex ROA column (300×7.8 mm, Phenomenex, USA), detailed in section 3.2.7. All experiments were conducted in triplicates and the values have been represented as mean±standard error.
Fig. 4.3. Killing curve ofC. acetobutylicumMTCC 11274 when exposed to different duration of UV irradiation from a distance of 25 cm.
selected exposure time was 15 min, which resulted in killing of 90% of the cells (Fig.
4.4B). Similar to chemical mutagenesis using NTG, the concentrations and exposure time of EMS treatment were optimized. 90% killing of cells was observed at 16 g L-1 (Fig. 4.5A), which was selected for further optimization of exposure duration of cell to the mutagen. Optimum time required to kill 90% of the cells was found to be 30 min (Fig. 4.5B). The optimized conditions were then used to perform mutagenesis experiments followed by rational screening for butanol tolerant mutants.
Fig. 4.4. Killing curveC. acetobutylicumMTCC 11274 when exposed to different (A) NTG concentration (g L-1) and (B) exposure time (min).
Fig. 4.5. Killing curveC. acetobutylicumMTCC 11274 when exposed to different (A) EMS concentration (g L-1) and (B) exposure time (min).
Page|84 Department of Biosciences & Bioengineering| TH-2103_126106021
4.3.2 Rational screening of solvent tolerant mutant(s)
Evolutionary engineering comprising mutagenesis followed by selection has been widely applied to improve microbial tolerance to environmental stresses (Zhu et al., 2015). Keeping this view in mind, three mutagenesis experiments were carried out namely, UV irradiation, NTG treatment and mutagenesis using EMS, according to the optimum conditions obtained in section 4.3.1.
After the mutagenesis treatment, the untreated and treated cells were exposed to the selection pressure, which was 1.5% (w/v) butanol in the present study. A control experiment parallel to screening was also carried out where the untreated and treated cells were grown in absence of selection pressure. In case of UV irradiation, even after repeated trials the mutagenesis failed to generate any probable improvement in terms of solvent tolerance. Biomass growth was observed in the control experiments showing that the cells were able to survive the mutagenesis experiment; however, they were not mutated to tolerate high butanol concentration.
After a number of trials, NTG and EMS mutagenesis resulted in one and eight putatively improved strains, respectively. As reported in previous literature works, among the mutagens, EMS and NTG are the most effective ones and UV irradiation was not found to be an effective way to mutagenize solvent-producing Clostridia (Lemmel, 1984; Bowring and Morris, 1985). The results obtained in the present study corroborates with the findings of other researchers.
4.3.3 Characterization of putatively improved strain(s) for butanol tolerance and butanol production
The eight strains (E1-E8) selected after mutagenesis using EMS on the basis of improved butanol tolerance and the strain (M1) selected after mutagenesis using NTG, were characterized in batch fermentation to compare their growth, butanol titer, and butanol productivity in comparison to the wild type (WT) strain MTCC 11274. It was observed that despite having improved tolerance, none of the strains obtained after mutagenic treatment showed significant improvement in any of the desirable attributes such as biomass titer, butanol titer or butanol productivity (Fig.
4.6). A maximum butanol titer of 12.85 g L-1 was obtained by M1 which was similar to WT (12.3 g L-1). EMS and NTG treatments were repeated to get improved strains;
however, repeated trials did not yield any desirable strains. Therefore, further experiments were carried out using the wild type strain MTCC 11274.
4.3.4 Adaptive evolution to enhance butanol tolerance
Evolutionary engineering of cells via adaptation towards high butanol tolerance was carried out. Phase one of serial adaptation was started with exposure of the cells to 0.25% (w/v) butanol and incubating at 37°C for 24 h. This constituted one adaptation cycle and was carried out in the similar way till the stressed cells
showed growth similar to control (unstressed cells). After four-adaptation cycles at
Fig. 4.6. Bar chart depicting comparison of putatively improved strains after mutagenic treatment with wild type (WT) strain in terms of biomass (grey bar), butanol titer (back bar) and butanol productivity (red circle).
Fig. 4.7. Dynamic profile of biomass growth for cells exposed to different butanol concentration in comparison to unstressed cells. Control (•); cycle 1 (◦);
cycle 2 (È); cycle 3 (Í); cycle 4 (); cycle 5 (); cycle 6 (); cycle 7 (◊);
cycle 8 (Î).
0.25% (w/v) butanol, the cells exhibited similar biomass, specific growth rate, and lag time as the control and were considered to be adapted (Fig. 4.7). Further, second phase of adaptation was started with 0.5% (w/v) butanol and after five adaptation cycles, the strain was found to be adapted (Fig. 4.7). Similarly, the strain found to be adapted in the third phase of adaptation (exposure to 0.75% butanol) after seven cycles (Fig. 4.7). Adaptation to 1.0% was carried out for 18 cycles; however, no further improvement was achieved in terms of specific growth rate.