Zhong-Ce Hu

Biodegradation of aniline in an alkaline environment by a novel strain of the halophilic bacterium, Dietzia natronolimnaea JQ-AN.

Dietzia natronolimnaea JQ-AN was isolated from industrial wastewater containing aniline. Under aerobic conditions, the JQ-AN strain degraded 87% of the aniline in a 300 mg L(-1) aniline solution after 120 h of shake flask incubation in a medium containing sodium acetate. This strain had an unusually high salinity tolerance in minimal medium (0-6% NaCl, w/v). The optimal pH for microbial growth and aniline biodegradation was pH 8.0. Two liters of simulated aniline wastewater was created in a reactor at pH 8.0 and 3% NaCl (w/v), and biodegradation of aniline was tested over 7 days at 30 °C. For the initial concentrations of 100, 300, and 500 mg L(-1), 100%, 80.5% and 72% of the aniline was degraded, respectively. Strain JQ-AN may use an ortho-cleavage pathway for dissimilation of the catechol intermediate.

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor.

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6g/l yeast extract, 4.7 g/l glycerol, 42.1g/l mannitol, 0.5 g/l K(2)HPO(4), 0.5 g/l KH(2)PO(4), 0.1g/l MgSO(4)·7H(2)O, and 2.0 g/l CaCO(3) with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8±2.4% of conversion rate of glycerol to DHA was achieved after 72h of biotransformation using 10g/l resting cells at 30°C, pH 5.0 and 1.5vvm of aeration rate.

Nitrite-mediated synthesis of chiral epichlorohydrin using halohydrin dehalogenase from Agrobacterium radiobacter AD1

In the current study, the haloalcohol dehalogenase HheC gene from Agrobacterium radiobacter AD1 was synthesized and expressed in Escherichia coli. After purification using Ni–nitrilotriacetic acid affinity chromatography, HheC was used in the synthesis of chiral epichlorohydrin in the presence of NO2−. The optimal pH, temperature, and NO2− concentration for enantioselectivity are 5.0, 37°C, and 60 mM, respectively. The maximum velocity and Michaelis constant values for (S)‐epichlorohydrin are 714.3 µmol min−1 mg−1 and 17.2 mM, respectively, whereas those for (R)‐epichlorohydrin are 166.8 µmol min−1 mg−1 and 29.0 mM, respectively. Under optimal conditions, (R)‐epichlorohydrin with 99% enantiomeric excess was obtained after an 18 Min reaction; the yield reached 41%, which is the highest amount obtained for chiral epichlorohydrin synthesis using haloalcohol dehalogenase. In addition, (R)‐epichlorohydrin with 99% enantiomeric excess was successfully obtained from 1,3‐dichloro‐2‐propanol by the ring opening of racemic epichlorohydrin in the presence of NO2− after the ring closure of 1,3‐dichloro‐2‐propanol with HheC. To the best of our knowledge, the current study is the first report on the kinetic resolution of epichlorohydrin with NO2− and synthesis of chiral epichlorohydrin with 99% enantiomeric excess from 1,3‐dichloro‐2‐propanol by combining ring closure of 1,3‐dichloro‐2‐propanol and ring opening of racemic epichlorohydrin.

Biosynthesis of (R)-epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase

The substrate concentration and yield were shown to be very low in the production of (R)‐epichlorohydrin by hydrolysis of racemic epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)‐epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic epichlorohydrin was obtained. However, the ee of (R)‐epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)‐3‐chloro‐1,2‐propanediol displayed non‐competitive inhibition in the conversion of (S)‐epichlorohydrin but non‐significant inhibition for (R)‐epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)‐epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two‐phase reaction system, with a high yield (>27%) and ee (>98%) of (R)‐epichlorohydrin. This is the first report of high‐yield production of (R)‐epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.

Repeated biotransformation of glycerol to 1,3-dihydroxyacetone by immobilized cells of Gluconobacter oxydans with glycerol- and urea-feeding strategy in a bubble column bioreactor.

Some inorganic nitrogen sources and amino acids instead of yeast extract, which resulted in trouble of product purification, were introduced for 1,3-dihydroxyacetone (DHA) production by biotransformation with Gluconobacter oxydans. The results showed that urea is an optimal nitrogen source. Furthermore, the effects of glycerol- and urea-feeding strategies for DHA production by immobilized cells in a home-made bubble column bioreactor were optimized. Cells immobilization was prepared by cultivation in the bioreactor packed with porous ceramics, and then the broth was removed. Then, repeated biotransformation by continuous-feeding of glycerol and urea was developed. Up to 96.4±4.1g/L of average DHA concentration with 94.8±2.2% of average conversion rate of glycerol to DHA was achieved after 12 cycles of run. Near colorless DHA solution with few impurities was obtained and the production cost could be decreased.

IMPROVEMENT OF 1,3-DIHYDROXYACETONE PRODUCTION FROM Gluconobacter oxydans BY ION BEAM IMPLANTATION

Improvement of dihydroxyacetone (DHA) production by mutagenesis of ion beam implantation and medium optimization using response-surface methodology (RSM) were investigated in this work. More than 1000 mutant strains were selected through a mutagenesis method using N+ ions implantation with a dose of 60 × (2.6 × 1013) ions/cm2 and energy of 10 keV. Several high-yield mutant strains were showed the potent application for DHA production and the genetically stable mutant strain G. oxydans ZJB09113 was selected for optimization of cultivation condition by RSM. The optimal medium for DHA fermentation is composed (in g/L) of yeast extract 4.88, CaCO3 2.00, and glycerol 52.86 mL/L (initial pH 4.89). The maximal DHA concentration of 40.0 g/L was achieved after 24 hr of shaken flask fermentation at 30°C with 150 rpm, and 196.3% increase in DHA production in comparison with unoptimized conditions.

Enhancement of epoxide hydrolase production by 60Co gamma and UV irradiation mutagenesis of Aspergillus niger ZJB‐09103

An effective epoxide hydrolase (EH) production strain was mutagenized using 60Co gamma and UV irradiation. Among positive mutant strains, the EH activity of C2‐44 reached 33.7 U/g, which was 267% as much as that of the original Aspergillus niger ZJB‐09103. Compared with the wild type, there were significant changes in morphology for C2‐44, including the color of mycelia on the slants and the shape of conidial head. In addition, glucose and soybean cake were the optimal carbon and nitrogen source in terms of EH activity for the mutant C2‐44 instead of soluble starch and peptone for the wild‐type strain. The reaction time required to reach 99% enantiomeric excesses of (S)‐epichlorohydrin from racemic substrate was shortened significantly by the mutant C2‐44. This phenomenon was probably explained by the higher Vmax for hydrolysis of racemic epichlorohydrin by C2‐44 compared with Aspergillus niger ZJB‐09103.

Biodegradation of m-cresol in alkaline wastewater by resting photosynthetic bacteria

AbstractThe application of various micro-organisms to metabolize organic compounds is one potentially effective method in treating hazardous and toxic wastewater. The effects of doses of cells, pH, initial substrate concentrations, and the addition of metal ions (Mn2+ and Cu2+) on m-cresol biodegradation by the resting cells (non-growth cells without immobilizing) of photosynthetic bacteria (PSBr) are investigated on a rotary shaker at 120 rpm at 30°C. The maximum biodegradation rate for a 200 mg/L m-cresol solution was 98% with a PSBr dose of 0.17 g/L, pH 7.5 at 30°C in dark conditions. The degradation capability of PSBr was drastically reduced in the presence of Mn2+ or Cu2+, and the inhibitory effect of Cu2+ was greater than that of Mn2+ for the same concentration. Specific degradation rates were fitted with the Haldane kinetic model. The parameter values were; rmax = 0.05/h, Km = 254.15 mg/L, and Ki = 112.18 mg/L, with a coefficient of determination (R2) greater than 0.89. Our results suggest that PSB...