Yucai He

Enhancement of enzymatic saccharification of corn stover with sequential Fenton pretreatment and dilute NaOH extraction

In this study, an effective method by the sequential Fenton pretreatment and dilute NaOH extraction (FT-AE) was chosen for pretreating corn stover. Before dilute NaOH (0.75 wt%) extraction at 90 °C for 1h, Fenton reagent (0.95 g/L of FeSO4 and 29.8 g/L of H2O2) was employed to pretreat CS at a solid/liquid ratio of 1/20 (w/w) at 35 °C for 30 min. The changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated solid residue were correlated with the enhancement of enzymatic saccharification. After being enzymatically hydrolyzed for 72 h, the reducing sugars and glucose from the hydrolysis of 60 g/L FT-AE-CS pretreated could be obtained at 40.96 and 23.61 g/L, respectively. Finally, the recovered hydrolyzates containing glucose had no inhibitory effects on the ethanol fermenting microorganism. In conclusion, the sequential Fenton pretreatment and dilute NaOH extraction has high potential application in future.

Enzymatic saccharification of sugarcane bagasse by N-methylmorpholine-N-oxide-tolerant cellulase from a newly isolated Galactomyces sp. CCZU11-1

Based on the enrichment culture strategy, a novel N-methylmorpholine-N-oxide (NMMO)-tolerant cellulase-producing strain Galactomyces sp. CCZU11-1 was isolated from soil samples. After the optimization of culture condition, the highest FPA (13.4 U/mL) and CMCase (24.5 U/mL) were obtained. In both culture and reaction media containing NMMO 25% (w/v), the cellulase from Galactomyces sp. CCZU11-1 still had good activity. Furthermore, high saccharification rate was obtained in aqueous-NMMO media. Moreover, the fermentability of the hydrolyzates, obtained after enzymatic in situ saccharification of the NMMO-pretreated sugarcane bagasse, was evaluated using Saccharomyce scerevisiae. In conclusion, Galactomyces sp. CCZU11-1 is a promising candidate as high NMMO-tolerant cellulase producer and has potential application in future.

Improving enzymatic saccharification of bamboo shoot shell by alkalic salt pretreatment with H2O2

Pretreatment of bamboo shoot shell (BSS) by a combination of alkalic salts with hydrogen peroxide (H2O2) was evaluated for its delignification effect and for its ability to enhance enzymatic saccharification of pretreated solids. By comparing different alkalic salts, the combination of 9% Na3PO4·12H2O and 0.3g/g H2O2 (ASHP) was identified as an effective system that showed the highest delignification of 87.7% and the total reducing sugar yield of 97.1% when pretreated BSS at a solid to liquid ratio of 1/20 (w/w) at 80°C for 2h. The delignification effect and the disruption of the lignocelluloses structure by this novel pretreatment method were deduced to be the main reasons that led to enhanced enzymatic saccharification as supported by the chemical composition analysis and the results of SEM, FTIR and XRD analyses of the untreated and alkalic salt pretreated BSS.

A combined sodium phosphate and sodium sulfide pretreatment for enhanced enzymatic digestibility and delignification of corn stover

Na3PO4 and Na2S were employed as efficient alkaline catalysts for the pretreatment of corn stover. To systematically obtain optimal conditions, the effects of critical pretreatment parameters including sodium phosphate concentration (1-4%), sulfidity (0-20%), pretreatment temperature (100-120°C), and reaction time (20-60min) on the reducing sugar yield of pretreated substrates were evaluated in a lab-scale using the response surface methodology. Pretreated under the sodium phosphate concentration of 4%, sulfidity of 10%, temperature of 120°C, and reaction time of 40min, the reducing sugar yield and glucose yield of the pretreated corn stover achieved 91.11% and 64.01%, respectively, with a moderate enzyme loading of 30FPU/g substrate. Additionally, a strong correlation (R(2)=0.971 and R(2)=0.954) between the delignification and the reducing sugar yield (or glucose yield) was observed by this pretreatment method. These results evidently support that the combined Na3PO4-Na2S pretreatment is an effective and feasible method for processing lignocellulosic biomass.

Enzymatic in situ saccharification of chestnut shell with high ionic liquid-tolerant cellulases from Galactomyces sp. CCZU11-1 in a biocompatible ionic liquid-cellulase media.

In this study, it was the first time to report that the cellulases of Galactomyces sp. CCZU11-1 showed high activity and stability in the culture and reaction media containing IL [Mmim]DMP. Using untreated chestnut shell (CNS) as carbon source in the culture media containing IL [Mmim]DMP (5%, w/v), high activity of FPA (28.6U/mL), xylanase (186.2U/mL), and CMCase (107.3U/mL) were obtained, and 184.9mg/L of total protein was achieved. Furthermore, the changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS utilized with Galactomyces sp. CCZU11-1 were characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. After was enzymatically hydrolyzed with the prepared crude enzymes in IL diluted to 20% (w/v), a high yield of reducing sugars, 62.1%, was obtained. Significantly, Galactomyces sp. CCZU11-1 showed high potential for the efficient transformation of lignocellulosic materials to glucose in a single-step process.

Mild alkaline presoaking and organosolv pretreatment of corn stover and their impacts on corn stover composition, structure, and digestibility

An efficient strategy was developed in current work for biochemical conversion of carbohydrates of corn stover into monosaccharides. Corn stover was first presoaked in mild alkaline solution (1% Na2S) under 40°C for 4h, after which about 35.3% of the lignin was successfully removed while the specific surface area was notably enlarged. Then the presoaked solids were subjected to organosolv pretreatment that employed 20% methanol with an addition of 0.2% HCl as catalyst at 160°C for 20min, and the maximum total sugar yield of the pretreated corn stover achieved was 98.6%. The intact structure of corn stover was disrupted by this two-step process, which resulted in a porous but crystalline structure of the regenerated solids that were mainly composed of cellulose. The enlarged specific surface area and increased accessibility made the regenerated solids highly digestible by a moderate enzyme loading.

Effective asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate by recombinant E. coli CCZU-A13 in [Bmim]PF6-hydrolyzate media.

It was the first report that the concentrated hydrolyzates from the enzymatic hydrolysis of dilute NaOH (3wt%)-soaking rice straw at 30°C was used to form [Bmim]PF6-hydrolyzate (50:50, v/v) media for bioconverting ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] (>99% e.e.) with recombinant E. coli CCZU-A13. Compared with pure glucose, the hydrolyzates could promote both initial reaction rate and the intracellular NADH content. Furthermore, emulsifier OP-10 (20mM) was employed to improve the reductase activity. Moreover, Hp-β-cyclodextrin (0.01mol Hp-β-cyclodextrin/mol COBE) was also added into this bioreaction system for enhancing the biosynthesis of (R)-CHBE from COBE by E. coli CCZU-A13 whole-cells. The yield of (R)-CHBE (>99% e.e.) from 800mM COBE was obtained at 100% in the [Bmim]PF6-hydrolyzate (50:50, v/v) media by supplementation of OP-10 (20mM) and Hp-β-CD (8mM). In conclusion, an effective strategy for the biosynthesis of (R)-CHBE was successfully demonstrated.

Discovery of a reductase-producing strain recombinant E. coli CCZU-A13 using colorimetric screening and its whole cell-catalyzed biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate.

An NADH-dependent reductase (SsCR) was discovered by genome data mining. After SsCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-A13 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) was screened using one high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (R)-CHBE (>99% ee) with the resting cells of E. coli CCZU-A13 was successfully demonstrated in n-butyl acetate-water (10:90, v/v) biphasic system. Biotransformation of 600mM COBE for 8h in the biphasic system, (R)-CHBE (>99% ee) could be obtained in the high yield of 100%. Moreover, the broad substrate specificity in the reduction of aliphatic and aromatic carbonyl compounds was also found. Significantly, E. coli CCZU-A13 shows high potential in the industrial production of (R)-CHBE (>99% ee) and its derivatives.

Biological conversion of the aqueous wastes from hydrothermal liquefaction of algae and pine wood by Rhodococci.

In this study, R. opacus PD630, R. jostii RHA1, R. jostii RHA1 VanA-, and their co-culture were employed to convert hydrothermal liquefaction aqueous waste (HTLAW) into lipids. After 11days, the COD reduction of algal-HTLAW reached 93.4% and 92.7% by R. jostii RHA1 and its mutant VanA-, respectively. Woody-HTLAW promoted lipid accumulation of 0.43glipid/gcell dry weight in R. opacus PD630 cells. Additionally, the total number of chemicals in HTLAW decreased by over 1/3 after 7days of coculture, and 0.10g/L and 0.46g/L lipids were incrementally accumulated in the cellular mass during the fermentation of wood- and algal-HTLAW, respectively. The GC-MS data supported that different metabolism pathways were followed when these Rhodococci strains degraded algae- and woody-HTLAW. These results indicated promising potential of bioconversion of under-utilized carbon and toxic compounds in HTLAW into useful products by selected Rhodococci.

Enhanced bioreduction synthesis of ethyl ( R )-4-chloro-3-hydroybutanoate by alkalic salt pretreatment

In this study, biomass-hydrolysate was used for enhancing the bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE). Firstly, dilute alkalic salt pretreatment was attempted to pretreat bamboo shoot shell (BSS). It was found that enzymatic in situ hydrolysis of 20-50 g/L BSS pretreated with dilute alkalic salts (0.4% Na2CO3, 0.032% Na2S) at 7.5% sulfidity by autoclaving at 110 °C for 40 min gave sugar yields at 59.9%-73.5%. Moreover, linear relationships were corrected on solid recovery-total delignification-sugar yield. In BSS-hydrolysates, xylose and glucose could promote the reductase activity of recombinant E. coli CCZU-A13. Compared with glucose, hydrolysate could increase the reductase activity by 1.35-folds. Furthermore, the cyclohexane-hydrolysate (10:90, v/v) biphasic media containing ethylene diamine tetraacetic acid (EDTA, 40 mM) and l-glutamine (150 mM) was built for the effective biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] (94.6% yield) from 500 mM COBE. In conclusion, this strategy has high potential for the effective biosynthesis of (R)-CHBE (>99% e.e.).