Yilan Liu

Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value.

Succinic acid is an important precursor for the synthesis of high-value-added products. Saccharomyces cerevisiae is a suitable platform for succinic acid production because of its high tolerance towards acidity. In this study, a modified pathway for succinate production was established and investigated in S. cerevisiae. The engineered strain could produce up to 6.17±0.34g/L of succinate through the constructed pathway. The succinate titer was further improved to 8.09±0.28g/L by the deletion of GPD1 and even higher to 9.98±0.23g/L with a yield of 0.32mol/mol glucose through regulation of biotin and urea levels. Under optimal supplemental CO2 conditions in a bioreactor, the engineered strain produced 12.97±0.42g/L succinate with a yield of 0.21mol/mol glucose at pH 3.8. These results demonstrated that the proposed engineering strategy was efficient for succinic acid production at low pH value.

Novel membrane-based biotechnological alternative process for succinic acid production and chemical synthesis of bio-based poly (butylene succinate)

Succinic acid was produced in a novel membrane-based fermentation and separation integrated system. With this integrated system, product inhibition was alleviated by removing acids and replenishing fresh broth. High cell density maintain for a longer time from 75 to 130h and succinic acid concentration increased from 53 to 73g/L. In the developed separation process, succinic acid was crystallized at a recovery of 85-90%. The purity of the obtained succinic acid crystals reached 99.4% as found by HPLC and (1)H NMR analysis. A crystallization experiment indicated that among by-products glucose had a negative effect on succinic acid crystallization. Poly (butylene succinate) (PBS) was synthesized using the purified succinic acid and (1)H NMR analysis confirmed that the composition of the synthesized PBS is in agreement with that from petro-based succinic acid.

Efficient production of succinic acid from macroalgae hydrolysate by metabolically engineered Escherichia coli

In this study, microbial production of succinic acid from macroalgae (i.e., Laminaria japonica) was investigated for the first time. The engineered Escherichia coli BS002 exhibited higher molar yield of succinic acid on mannitol (1.39±0.01mol/mol) than glucose (1.01±0.05mol/mol). After pretreatment and enzymatic hydrolysis, L. japonica hydrolysate was mainly glucose (10.31±0.32g/L) and mannitol (10.12±0.17g/L), which was used as the substrate for succinic acid fermentation with the recombinant BS002. A final 17.44±0.54g/L succinic acid was obtained from the hydrolysate after 72h dual-phase fermentation. The yield was as high as 1.24±0.08mol/mol total sugar, which reached 73% of the maximum theoretical yield. The results demonstrate that macroalgae biomass represents a novelty and economical alternative feedstock for biochemicals production.

Performance of a haloalkaliphilic bioreactor and bacterial community shifts under different COD/SO42- ratios and hydraulic retention times

Sulfur dioxide from flue gas was converted into sulfate after the absorption of alkaline solutions. Haloalkaliphilic microorganisms have been used in reducing sulfate to decrease expenses and avoid sulfide inhibition. The effects of different COD/SO4(2-) ratios and hydraulic retention times (HRTs) on the sulfate removal efficiency and bacterial community were investigated in model experiments. Ethanol showed better performance as an electron donor than lactate. The optimum COD/SO4(2-) ratio and HRT were 4.0 and 18 h, respectively, with respective sulfate removal efficiency and rate of 97.8 ± 1.11% and 6.26 ± 0.0710 g/Ld. Sulfide concentrations reached 1,603 ± 3.38 mg/L. Based on denaturing gradient gel electrophoresis analysis of 16S rDNA, the major sulfate-reducing bacterium (SRB) was Desulfonatronovibrio sp., which was only detected at a COD/SO4(2-) ratio of 4.0 using ethanol as an electron donor. Different HRTs had no significant effect on the band corresponding to this species. PCR results show that methane-producing archaea (MPA) were from the acetoclastic methanogenic family Methanosarcinaceae. Quantitative real-time PCR did not demonstrate any significant competition between SRB and MPA. The findings of this study indicate that sulfate reduction, nitrate reduction, and sulfide oxidization may occur in the same bioreactor.

Performance of a haloalkaliphilic bioreactor under different NO3-/SO42- ratios

Effects of NO3(-)/SO4(2-) ratio on denitrification and sulfate removal efficiency were investigated in model experiments applying haloalkaliphilic bioreactor. The reduction of both substrates performed well at different NO3(-)/SO4(2-) ratios ranging from 17.6 to l.5. The removal rates of nitrate and sulfate were 6 and 1.39kgm(-3)d(-1), respectively, at NO3(-)/SO4(2-) ratio 3.0, while sulfide concentration reached up to 703gm(-3). The major sulfate-reducing and denitrifying bacteria were Desulfonatronovibrio sp. and Halomonas campisalis, respectively. Decrease in NO3(-)/SO4(2-) ratio led to obvious changes in bacterial community. Although the sulfate reducers became dominant, the population of denitrifying ones also increased as it was demonstrated by analysis of PCR-amplified 16S rDNA fragments, which suggested that SRB and DB coexisted well in bioreactor.

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.

Analysis of mixtures of fatty acids and fatty alcohols in fermentation broth

Microbial production of fatty acids and fatty alcohols has attracted increasing concerns because of energy crisis and environmental impact of fossil fuels. Therefore, simple and efficient methods for the extraction and quantification of these compounds become necessary. In this study, a high-performance liquid chromatography-refractive index detection (HPLC-RID) method was developed for the simultaneous quantification of fatty acids and fatty alcohols in these samples. The optimum chromatographic conditions are C18 column eluted with methanol:water:acetic acid (90:9.9:0.1, v/v/v); column temperature, 26°C; flow rate, 1.0mL/min. Calibration curves of all selected analytes showed good linearity (r(2)≥0.9989). The intra-day and inter-day relative standard deviations (RSDs) of the 10 compounds were less than 4.46% and 5.38%, respectively, which indicated that the method had good repeatability and precision. Besides, a method for simultaneous extraction of fatty acids and fatty alcohols from fermentation broth was optimized by orthogonal design. The optimal extraction conditions were as follows: solvent, ethyl acetate; solvent to sample ratio, 0.5:1; rotation speed, 2min at 260rpm; extraction temperature, 10°C. This study provides simple and fast methods to simultaneously extract and quantify fatty acids and fatty alcohols for the first time. It will be useful for the study of microbial production of these products.