Screening of butanol producing strain with high phenolic acid tolerance by the approach of multi-repeating stress acclimation

Carbon-neutral fuels (such as ethanol and butanol) have gradually drawn much attention in recent years, due to the ever-increasing perception of global warming and environmental protection. Among them, butanol can be expected to serve as one of the promising candidates for biofuels, such as less corrosive, higher octane number, lower solubility in water, and higher energy content, compared with ethanol. However, the high feedstock cost and low productivity can be still a challenge in the acetone-butanol-ethanol (ABE) fermentation, due to the product's (especially butanol) resistance or toxicity to the current butanol-producing strains. The overall economics of bio-butanol production can be enhanced using the abundant source, and low-price of materials (such as lignocellulose). Nevertheless, it must be appropriately pretreated, when these materials are used as the feedstock for the ABE fermentation. Various compounds can be formed during pretreatment. Moreover, the phenolic acids that are derived from lignin degradation can be the most toxicity inhibitor for the butanol-producing strains. In this study, some excellent strains were screened for the high tolerant phenolic acids stress and high butanol production. A multi-factor screening strategy was carried out to obtain the strain W6, in order to synthesize the sufficient reducing power and high tolerance to butanol. Furthermore, the strain W6 was then domesticated to further enhance the butanol tolerance via the appropriate concentration of butanol stress. As such, strain W6-1 was obtained, where the butanol and total solvent production were (8.22 ± 0.21) and (11.72 ± 0.26) g/L, respectively, which were 14.01% and 16.85% higher than that of strain W6. After that, strain W6-1 was treated to improve the production of butanol using UV mutagenesis combined with the multi-factor screening model. The high butanol production strain W6-2 was then selected after treatment. The butanol and total solvent production reached (9.51 ± 0.06) and (15.32 ± 0.11) g/L, resulting in an increase of 15.69% and 30.72%, respectively, compared with the strain W6-1. Finally, the high phenolic acid-tolerant strain W6-3 was achieved by the adaptive evolution strategy with phenolic acid stress condition. At the end of fermentation, the biomass increased by 50.00%, whereas, the butanol and solvent production of the mutant strain W6-3 increased by 18.17% and 17.49%, respectively, compared with the strain W6-2. When strain W6-3 was in the 0.5 g/L of phenol acid stress environment, the butanol production were 12.11% higher than that of strain W6-2, respectively. Once the phenolic acid stress concentration reached 1.0 g/L, there was no growth in the pre-domestication strain W6-2. However, the total solvent production of (3.42 ± 0.42) g/L was obtained for the mutant strain W6-3, indicating excellent phenol acid tolerance. Taking the puerariae slag hydrolysate as the fermentation feedstock, the production of butanol was (8.54 ± 0.31) g/L in the mutant strains W6-3, which was 26.71% higher than that of the strain W6-2. The phenolic acid tolerance and fermentation performance of the mutant strain were greatly improved after multiple rounds of mutagenesis and adaptive evolution. This finding can provide a reliable theoretical reference to rapidly screen the excellent producing trains, in order to fully meet the requirements of fermentation performance.