Lipid synthesis by an acidic acid tolerant Rhodotorula glutinis / 生物工程学报

Acetic acid, as a main by-product generated in the pretreatment process of lignocellulose hydrolysis, significantly affects cell growth and lipid synthesis of oleaginous microorganisms. Therefore, we studied the tolerance of Rhodotorula glutinis to acetic acid and its lipid synthesis from substrate containing acetic acid. In the mixed sugar medium containing 6 g/L glucose and 44 g/L xylose, and supplemented with acetic acid, the cell growth was not:inhibited when the acetic acid concentration was below 10 g/L. Compared with the control, the biomass, lipid concentration and lipid content of R. glutinis increased 21.5%, 171% and 122% respectively when acetic acid concentration was 10 g/L. Furthermore, R. glutinis could accumulate lipid with acetate as the sole carbon source. Lipid concentration and lipid yield reached 3.20 g/L and 13% respectively with the initial acetic acid concentration of 25 g/L. The lipid composition was analyzed by gas chromatograph. The main composition of lipid produced with acetic acid was palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, including 40.9% saturated fatty acids and 59.1% unsaturated fatty acids. The lipid composition was similar to that of plant oil, indicating that lipid from oleaginous yeast R. glutinis had potential as the feedstock of biodiesel production. These results demonstrated that a certain concentration of acetic acid need not to be removed in the detoxification process when using lignocelluloses hydrolysate to produce microbial lipid by R. glutinis.

Construction of polyhydroxybutyrate pathway in Klebsiella pneumoniae / 生物工程学报

1,3-propanediol production with the byproduct of biodiesel production is important to increase the economic benefit of biodiesel industry. Accumulation of 3-hydroxypropionaldehyde is one of the key problems in the 1,3-propanediol fermentation process, leading to the cell death and the fermentation abnormal ceasing. Different from the traditional way of reducing the accumulation of the 3-hydroxypropionaldehyde, we introduced the polyhydroxybutyrate pathway into the Klebsiella pneumoniae for the first time to enhance the tolerance of K. pneumoniae to 3-hydroxypropionaldehyde, at the same time, to improve the 1,3-propanediol production. Plasmid pDK containing phbC, phbA, phbB gene was constructed and transformed into K. pneumoniae successfully. PHB was detected in the engineered K. pneumoniae after IPTG induction and its content enhanced with the IPTG concentration increasing. The optimized IPTG concentration was 0.5 mmol/L. The constructed K. pneumoniae could produce 1,3-propanediol normally, at the same time accumulate polyhydroxybutyrate. With the constructed strain, the fermentation proceeds normally with the initial glucose was 70 g/L which the wild type strain stopped growing and the fermentation was ceasing; 1,3-propanediol concentration and yield reached 31.3 g/L and 43.9% at 72 h. Our work is helpful for the deep understanding of 1,3-propanediol metabolic mechanism of Klebsiella pneumoniae, and also provides a new way for strain optimization of Klebsiella pneumoniae.

Effect of acetic acid, furfural and 5-hydroxymethylfurfural on production of 2,3-butanediol by Klebsiella oxytoca / 生物工程学报

To get the tolerability and consumption of Klebsiella oxytoca on major inhibitors in lignocelluloses hydrolysate, we studied the effect of acetic acid, furfural and 5-hydroxymethylfurfural on production of 2,3-butanediol by Klebsiella oxytoca. The metabolites of furfural and 5-hydroxymethylfurfural were measured. The results show that when acetic acid, furfural and 5-hydroxymethylfurfural was individually added, tolerance threshold for Klebsiella oxytoca was 30 g/L, 4 g/L and 5 g/L, respectively. Acetic acid was likely used as substrate to produce 2,3-butanediol. The yield of 2,3-butanediol increased when acetic acid concentration was lower than 30 g/L. In the fermentation, more than 70% 5-hydroxymethylfurfural was converted to 2,5-furandimethanol. All furfural and the rest of 5-hydroxymethylfurfural were metabolized by Klebsiella oxytoca. It showed that in the detoxification process of 2,3-butanediol production using lignocelluloses hydrolysate, furfural should be given priority to remove and a certain concentration of acetic acid is not need to removal.

Pretreatment of oil palm residues by dilute alkali for cellulosic ethanol production / 生物工程学报

In the study, we used oil palm residues (empty fruit bunch, EFB) as raw material to produce cellulosic ethanol by pretreatment, enzymatic hydrolysis and fermentation. Firstly, the pretreatment of EFB with alkali, alkali/hydrogen peroxide and the effects on the components and enzymatic hydrolysis of cellulose were studied. The results show that dilute alkali was the suitable pretreatment method and the conditions were first to soak the substrate with 1% sodium hydroxide with a solid-liquid ratio of 1:10 at 40 degrees C for 24 h, and then subjected to 121 degrees C for 30 min. Under the conditions, EFB solid recovery was 74.09%, and glucan, xylan and lignin content were 44.08%, 25.74% and 13.89%, respectively. After separated with alkali solution, the pretreated EFB was washed and hydrolyzed for 72 h with 5% substrate concentration and 30 FPU/g dry mass (DM) enzyme loading, and the conversion of glucan and xylan reached 84.44% and 89.28%, respectively. We further investigated the effects of substrate concentration and enzyme loading on enzymatic hydrolysis and ethanol batch simultaneous saccharification and fermentation (SSF). The results show that when enzyme loading was 30 FPU/g DM and substrate concentration was increased from 5% to 25%, ethanol concentration were 9.76 g/L and 35.25 g/L after 72 h fermentation with Saccharomyces cerevisiae (inoculum size 5%, V/V), which was 79.09% and 56.96% of ethanol theory yield.

Key enzymes in butanol fermentation by a facultative anaerobe Bacillus sp. TSH1 / 生物工程学报

Bacillus sp. TSH1 is a butanol-producing microorganism newly isolated in our laboratory; it can grow and ferment under facultative anaerobic conditions, while sharing similar fermentation pathways and products with Clostridium acetobutylicum. To illustrate the relationships between the products and the enzyme activities in Bacillus sp. TSH1, key butanol- and ethanol-forming enzymes were studied, including butyraldehyde dehydrogenase, butanol dehydrogenase and alcohol dehydrogenase. The activities of the three enzymes increased rapidly after the initiation of fermentation. Activities of three enzymes peaked before 21 h, and simultaneously, product concentrations also began to increase gradually. The maximum activity of alcohol dehydrogenase was 0.054 U/mg at 12 h, butyraldehyde dehydrogenase 0.035 U/mg at 21 h and butanol dehydrogenase 0.055 U/mg at 15 h. The enzyme activities then decreased, but remained constant at a low level after 24 h, while the concentrations of butanol, acetone, and ethanol continued increasing until the end of the fermentation. The results will attribute to the understanding of the butanol metabolic mechanism, and provide a reference for further study of a facultative Bacillus metabolic pathway.

Kinetics of enzymatic hydrolysis of steam-explosion pretreated corn straw / 生物工程学报

In order to learn the enzymatic hydrolysis characteristics of steam-explosion pretreated corn straw by cellulase, the effects of substrate concentration, cellulase concentration and temperature were determined. The kinetics of the hydrolysis reaction could be described with the Michealis-Menten equation, and the hydrolysis reaction obeyed the classical first-order reaction rate in the first three hours. In the condition of 45 degrees C and pH 5.0 and the stirring rate 120 r/min, the Michealis constant (Km) and maximum rate (Vm) for 1.2 FPU/mL of cellulase were 11.71 g/L and 1.5 g/(L x h). The kinetic model, including the parameters such as substrate concentration, enzymatic concentration and temperature, was suit for the hydrolysis reaction under the temperature range from 30 degrees C-50 degrees C.

Optimization of ethylene production from ethanol dehydration using Zn-Mn-Co/HZSM-5 by response surface methodology / 生物工程学报

The effects of reaction temperature, ethanol concentration and weight hourly space velocity (WHSV) on the ethylene production from ethanol dehydration using zinc, manganese and cobalt modified HZSM-5 catalyst were investigated by response surface methodology (RSM). The results showed that the most significant effect among factors was reaction temperature and the factors had interaction. The optimum conditions were found as 34.4% ethanol concentration, 261.3 0 degrees C of reaction temperature and 1.18 h(-1) of WHSV, under these conditions the yield of ethylene achieved 98.69%.