Chapter 4. Enhancement of butanol tolerance of the selected strain via
4.2 Materials and methods
4.2.1 Microorganism, inoculum, and media
The lyophilized culture of best butanol producerClostridium acetobutylicum MTCC 11274 was revived and stored as glycerol stocks at 80°C. Inoculum preparation was carried out in tryptone-yeast extract-glucose (TYG) medium as detailed in section 3.2.1. Adaption and mutagenesis experiments were carried out in the optimized
medium for maximization of butanol productivity comprising (in g L-1) glucose:
82.0, peptone: 49.0, K2HPO4: 0.50, KH2PO4: 0.50, MgSO4.7H2O: 0.46, MnSO4.H2O:
0.023, FeSO4.7H2O: 0.023, NaCl: 0.023, CH3COONH4: 2.2, para-amino-benzoic acid: 0.01, and biotin: 0.001. Experimental conditions and sampling were kept same as in section 3.2.2, unless otherwise mentioned. All the chemicals (AR grade) were purchased from Himedia, India unless otherwise mentioned.
4.2.2 UV irradiation of cultures and induction of mutants
The procedure followed for UV mutagenesis is schematically depicted in Fig 4.1. Batch fermentation was carried out till the cells reached exponential phase. 5 mL of fermentation broth was taken in a petri plate and placed below a UV germicidal light fixed in a chamber. The distance between UV lamp and culture plate was kept 25 cm. The UV irradiation was carried out at varying exposure times from 50 to 350 sec. A control experiment was carried out parallel to the irradiation experiments.
The experiments were carried out in triplicate and about 0.1 mL each from these exposed plates was inoculated in fresh medium and biomass (OD600 nm) was estimated after incubation made in dark at 37°C for 24 h. Killing curve was constructed by plotting the maximum absorbance (OD600 nm) against the time of exposure. The exposure time with 90% killing was selected and used for development of solvent tolerant strain. The killing percentage was calculated by the following formulae:
Killing percentage (%)=(OD600Control−OD600Treated)
OD600Control ×100 (4.1)
The mutagenic experiments were carried out in triplicate and 0.1 mL from each of the three aliquots was inoculated in fresh medium for subsequent screening experiments.
Fig. 4.1. Schematic representation of UV mutagenesis protocol.
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4.2.3 Chemical mutagenesis using N-methyl-N-nitro-N-nitrosoguanidine (NTG)
Schematic representation of mutagenesis protocol is shown in Fig. 4.2. 2 mL exponential phase cells (OD600nm 3.0) were centrifuged at 5,000 rpm for 5 min. The pellet was re-suspended in fresh media containing different concentration of NTG solution (0.1 - 1.2 g L-1). After 15 min of incubation at 37°C, 1 mL of 2.5% (w/v) sodium thiosulphate was added to terminate the reaction. Cells were washed twice with fresh medium. 200µL from each eppendorf tube were inoculated in fresh media and incubated at 37°C. A control experiment was performed parallel to mutagenesis.
After 24 h incubation, the biomass (OD600 nm) was calculated and killing curve was constructed by plotting the maximum absorbance (OD600nm) against the mutagen concentration (Fig. 4.2A). The concentration with 90% killing, calculated according
Fig. 4.2. Schematic representation of chemical mutagenesis protocol.
to Eq. 4.1, was selected for further optimization of incubation time (5-40 min) of mutation treatment (Fig. 4.2B). Similarly, killing curve was constructed to select the duration for mutagen exposure. The optimized mutagen concentration and treatment time were used for development of the solvent tolerant strain.
4.2.4 Chemical mutagenesis using ethyl methane sulfonate (EMS)
The mutagenesis experiments were carried out similar to as described in section 4.2.3. The EMS concentration used were varied from 0.1-30 g L-1and initial exposure time was kept at 30 min. A control experiment was performed parallel to mutagenesis. The concentration chosen on the basis of 90% killing (calculated according to Eq. 4.1) was selected for further optimization of mutagen exposure
time (10-60 min) of mutation treatment. The optimized mutagen concentration and treatment time were used for development of the solvent tolerant strain.
4.2.5 Rational screening of solvent tolerant mutant(s)
All the mutagenesis experiments (UV, NTG, and EMS) described in sections 4.2.2-4.2.4 were performed in triplicate along with control. After mutagenic treatment, the cells were re-suspended in fresh media and cultured in micro titer plates containing different concentration of selection pressure (butanol). The wells exhibiting growth after incubation were further inoculated in tubes with medium containing selection pressure for second round of screening. The tubes showing growth were used for glycerol stock preparation and stored at -80°C for further characterization in terms of butanol tolerance and butanol production.
4.2.6 Characterization of mutants for butanol tolerance and butanol produc- tion
The stored glycerol stocks were revived and inoculum preparation was carried out as explained in section 4.2.1. All the characterization experiments were conducted in 500 mL customized cultivation bottles under static condition in an incubator at 37°C. Media preparation and experimental procedure were kept same as in section 3.2.2. The cultures were characterized on the basis of growth, pH, glucose utilization, and butanol production. A control experiment was carried out to compare performance of tolerant strains with the wild type strain. Mutants were also characterized in terms of their butanol tolerance and butanol challenge experiments were carried out as detailed in section 3.2.4.
4.2.7 Adaptive evolution to enhance butanol tolerance
While growing in nature bacterial cells encounters various environmental stresses which negatively alter their growth. One such stress is accumulation of large amount of toxic product, which is termed as product inhibition. However, the cells are also known to exhibit a process of adaptation whereby they gradually get accustomed to survive in harsh conditions also known as adaptive evolution. Keeping this in mind, a set of experiments were carried out to adapt the wild typeC. acetobutylicum MTCC 11274 to higher concentration of butanol. Inoculum preparation was carried out as mentioned earlier.
Exponentially growing cells were inoculated in two culture bottles containing butanol concentration of 0 (control) and 0.25 g L-1. The batch was monitored for 72 h for dynamic profiling of growth and butanol production. In addition, after reaching biomass of 1.0 g L-1(OD600 nm 3.0) 10% (v/v) of the cells were subcultured in fresh media having same butanol concentration as before and similar analysis was carried out. This process constituted one phase of adaptation and individual subcultures in each phase were termed as adaptation cycles. Several cycles were Page|82 Department of Biosciences & Bioengineering| TH-2103_126106021
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.