Hanjie Ying

Succinic acid production by Actinobacillus succinogenes using hydrolysates of spent yeast cells and corn fiber

The enzymatic hydrolysate of spent yeast cells was evaluated as a nitrogen source for succinic acid production by Actinobacillus succinogenes NJ113, using corn fiber hydrolysate as a carbon source. When spent yeast cell hydrolysate was used directly as a nitrogen source, a maximum succinic acid concentration of 35.5 g/l was obtained from a glucose concentration of 50 g/l, with a glucose utilization of 95.2%. Supplementation with individual vitamins showed that biotin was the most likely factor to be limiting for succinic acid production with spent yeast cell hydrolysate. After supplementing spent yeast cell hydrolysate and 90 g/l of glucose with 150 μg/l of biotin, cell growth increased 32.5%, glucose utilization increased 37.6%, and succinic acid concentration was enhanced 49.0%. As a result, when biotin-supplemented spent yeast cell hydrolysate was used with corn fiber hydrolysate, a succinic acid yield of 67.7% was obtained from 70.3 g/l of total sugar concentration, with a productivity of 0.63 g/(l h). Our results suggest that biotin-supplemented spent yeast cell hydrolysate may be an alternative nitrogen source for the efficient production of succinic acid by A. succinogenes NJ113, using renewable resources.

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.

Enhanced butanol production by modulation of electron flow in Clostridium acetobutylicum B3 immobilized by surface adsorption

The objective of this study was to improve butanol yield and productivity by redox modulation and immobilization of Clostridium acetobutylicum B3 cells. Stoichiometric network analysis revealed that NAD(P)H that had escaped from the fermentation as H2 limited the butanol yield and led to the accumulation of oxidation byproducts, e.g., acetone. Methyl viologen was used as an electron carrier to divert the electron flow away from H2 production and to reinforce the NAD(P)H supply. Butanol yield was increased by 37.8% with severely diminished acetone production. Immobilization of the cells by adsorption onto a fibrous matrix improved their butanol tolerance and production rate. An average of 15.6 g/L butanol was achieved within 12 h with a solvent productivity of 1.88 g/L/h in repeated batch fermentation. To our knowledge, this is the highest solvent productivity with a relatively high butanol titer produced by a Clostridium strain in batch fermentation.

Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate

Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium. An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine-Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to (S)-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles (S)-CHBE formed per mole NADP(+), was 12,600, which was more than 10-fold higher than in aqueous systems.

A new member of the short-chain dehydrogenases/reductases superfamily: Purification, characterization and substrate specificity of a recombinant carbonyl reductase from Pichia stipitis

A novel short-chain dehydrogenases/reductases superfamily (SDRs) reductase (PsCR) from Pichia stipitis that produced ethyl (S)-4-chloro-3-hydroxybutanoate with greater than 99% enantiomeric excess, was purified to homogeneity using fractional ammonium sulfate precipitation followed by DEAE-Sepharose chromatography. The enzyme purified from recombinant Escherichia coli had a molecular mass of about 35 kDa on SDS-PAGE and only required NADPH as an electron donor. The K(m) value of PsCR for ethyl 4-chloro-3-oxobutanoate was 4.9 mg/mL and the corresponding V(max) was 337 micromol/mg protein/min. The catalytic efficiency value was the highest ever reported for reductases from yeasts. Moreover, PsCR exhibited a medium-range substrate spectrum toward various keto and aldehyde compounds, i.e., ethyl-3-oxobutanoate with a chlorine substitution at the 2 or 4-position, or alpha,beta-diketones. In addition, the activity of the enzyme was strongly inhibited by SDS and beta-mercaptoethanol, but not by ethylene diamine tetra acetic acid.

Selective separation of biobutanol from acetone-butanol-ethanol fermentation broth by means of sorption methodology based on a novel macroporous resin.

The traditional distillation method for recovery of butanol from fermentation broth is an energy‐intensive process. Separation of butanol based on adsorption methodology has advantages in terms of biocompatibility and stability, as well as economy, and therefore gains much attention. However, the application of the commercial adsorbents in the integrated acetone–butanol–ethanol (ABE) fermentation process is restricted due to the low recovery (less than 85%) and the weak capability of enrichment in the eluent (3–4 times). In this study, we investigated the sorption properties of butanol onto three kinds of adsorbents with different polarities developed in our laboratory, that is, XD‐41, H‐511, and KA‐I resin. The sorption behaviors of single component and ABE ternary mixtures presented in the fermentation broths on KA‐I resin were investigated. KA‐I resin had higher affinity for butanol than for acetone, ethanol, glucose, acetic acid, and butyric acid. Multicomponent ABE sorption on KA‐I resin was modeled using a single site extended Langmuir isotherm model. In a desorption study, all the adsorbed components were desorbed in one bed volume of methanol, and the recovery of butanol from KA‐I resin was 99.7%. The concentration of butanol in the eluent was increased by a factor of 6.13. In addition, KA‐I resin was successfully regenerated by two bed volumes of water. Because of its quick sorption, high sorption capacity, low cost, and ease of desorption and regeneration, KA‐I resin exhibits good potential for compatibility with future ABE fermentation coupled with in situ recovery product removal techniques.

New potential inhibitors of DNA topoisomerase. Part II: Design and synthesis of α-lapachone derivatives under microwave irradiation

A new series of potential inhibitors of DNA topoisomerase II were synthesized from facile materials (aromatic aldehydes, Meldrums acid and 2-hydroxynaphthalene-1,4-dione) under microwave irradiation. The method provides a valuable tool in designing new and more potent cytotoxic analogues. This procedure is advantageous both economically and environmentally.

Design, synthesis, and antibiofilm activity of 2-arylimino-3-aryl-thiazolidine-4-ones.

A series of novel 2-arylimino-3-aryl-thiazolidine-4-ones was designed, synthesized and tested for in vitro antibiofilm activity against Staphylococcus epidermidis. Among them tested, some compounds with carboxylic acid groups showed good antibiofilm activity. The antibiofilm concentration of 1x was 6.25 microM. The structure-activity relationships revealed that incorporation of 2-phenylfuran moiety could greatly enhance antibiofilm activity of thiazolidine-4-one.

Enhancement of n-butanol production by in situ butanol removal using permeating-heating-gas stripping in acetone-butanol-ethanol fermentation.

Butanol recovery from acetone-butanol-ethanol (ABE) fed-batch fermentation using permeating-heating-gas was determined in this study. Fermentation was performed with Clostridium acetobutylicum B3 in a fibrous bed bioreactor and permeating-heating-gas stripping was used to eliminate substrate and product inhibition, which normally restrict ABE production and sugar utilization to below 20 g/L and 60 g/L, respectively. In batch fermentation (without permeating-heating-gas stripping), C. acetobutylicum B3 utilized 60 g/L glucose and produced 19.9 g/L ABE and 12 g/L butanol, while in the integrated process 290 g/L glucose was utilized and 106.27 g/L ABE and 66.09 g/L butanol were produced. The intermittent gas stripping process generated a highly concentrated condensate containing approximately 15% (w/v) butanol, 4% (w/v) acetone, a small amount of ethanol (<1%), and almost no acids, resulting in a highly concentrated butanol solution [∼ 70% (w/v)] after phase separation. Butanol removal by permeating-heating-gas stripping has potential for commercial ABE production.

Experimental and Modeling Studies on the Sorption Breakthrough Behaviors of Butanol from Aqueous Solution in a Fixed-bed of KA-I Resin

Removal of biobutanol from acetone-butanolethanol (ABE) fermentation broth can be achieved by fixed-bed sorption by means of KA-I resin, and the relevant breakthrough curves would provide much valuable information to help design a continuous fixed-bed sorption process in field application. In the present study, the effects of several important design parameters, i.e., initial butanol concentration (Cf: 3.0 ∼ 30.0 g/L), inlet flow rate (Qf: 0.5 ∼ 5.5 mL/min) and adsorbent bed height (Z: 4.2 ∼ 18.0 cm), on the adsorption breakthrough curves of KA-I resin in a fixed-bed column were investigated. It was found that the amount of adsorbed butanol at breakthrough point was increased with an increase in the value of Cf and Z; and with decrease in the value of Qf. However, the maximum sorption capacities of butanol at saturated point were basically unchanged. Three well-established fixed-bed adsorption models, namely Thomas, Yoon-Nelson and Adams-Bohart, were applied to predict the breakthrough curves and to determine the characteristic parameters of fixed-bed column, which are the basis for the process design at a real scale. Good agreement between the theoretical breakthrough curves and the experimental result were observed using Thomas and Yoon-Nelson models.