Maohua Yang

Efficient conversion of crop stalk wastes into succinic acid production by Actinobacillus succinogenes

Succinic acid is valued as a key platform chemical for use in a variety of synthetic applications. Efficient biosynthesis of succinic acid from renewable biomass resource is reported in this paper. Batch fermentations were carried out to analyze influence of several carbon sources on succinic acid production from feedstock wastes by Actinobacillus succinogenes BE-1. Crop stalk wastes, including corn stalk and cotton stalk, were enzymatically converted into a carbohydrate-rich feedstock, obtaining glucose concentrations approaching 65-80% of the total reducing sugar. For the anaerobic batch cultivation with cotton stalk hydrolysates, the production of succinic acid was 15.8 g l(-1) with a high yield of 1.23 g per g glucose. Glucose and xylose were utilized at same time, while cellubiose was not consumed until glucose and xylose were completely consumed.

High-concentration sugars production from corn stover based on combined pretreatments and fed-batch process.

In this paper, high-concentration sugars were produced from pretreated corn stover. The raw corn stover was pretreated in a process combining steam explosion and alkaline hydrogen-peroxide. The hemicellulose and lignin were removed greatly. The cellulose content increased to 73.2%. Fed-batch enzymatic hydrolysis was initiated with 12% (w/v) solids loading and 20 FPU/g solids. Then, 6% solids were fed consecutively at 12, 36 and 60 h. After 144 h, the final concentrations of reducing sugar, glucose, cellobiose and xylose reached 220, 175, 22 and 20 g/L, respectively. The final total biomass conversion was 60% in fed-batch process.

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.

Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130Z(T)

Succinic acid is one of the platform compounds and its production via natural feedstocks has drawn worldwide concerns. To evaluate the inhibitory effects of fermentation products on the growth of Actinobacillus succinogenes 130ZT and Escherichia coli NZN111, AFP111, BL21, fermentations with addition of individual products in medium were carried out. The cell growth was inhibited when the concentrations of formate, acetate, lactate, and succinate were at range of 8.8–17.6 g/L, 10–40 g/L, 9–18 g/L, and 10–80 g/L, respectively. For these two species of bacteria, E. coli was more resistant to acid products than A. succinogenes, while both endured succinate rather than by-products. As a result of end product inhibition, succinate production yield by A. succinogenes decreased from 1.11 to 0.49 g/g glucose. Logistic and Monod mathematical models were presented to simulate the inhibition kinetics. The Logistic model was found more suitable for describing the overall synergistic inhibitory effects.

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.

Complete genome sequence of Thialkalivibrio versutus D301 isolated from Soda Lake in northern China, a typical strain with great ability to oxidize sulfide

Thioalkalivibrio versutus D301 isolated from Soda Lake is a haloalkaliphilic and obligated chemolithoautotrophic Gram-negative bacterium. The strain has a good adaption to hyperhaline and highly alkaline environment and a powerful sulfur-oxidizing ability. Here, we present the complete genome sequence of T. versutus D301, providing insights into the genomic basis of its effects and facilitating its application in microbial desulfurization.