Enhanced butanol production by increasing NADH and ATP levels in Clostridium beijerinckii NCIMB 8052 by insertional inactivation of Cbei_4110.

Clostridium beijerinckii is identified as a promising Clostridium strain for industrialization of acetone and butanol (AB) fermentation. It has been reported that high reducing power levels are associated with high butanol yield. In this study, we regulated reducing power by blocking NAD(P)H consumption in C. beijerinckii NCIMB 8052. Gene Cbei_4110, encoding NADH-quinone oxidoreductase (nuoG), is a subunit of the electron transport chain complex I. After inactivation of gene Cbei_4110, the generated mutant strain exhibited a remarkable increase in glucose utilization ratio and enhanced butanol production to 9.5 g/L in P2 medium containing 30 g/L of glucose. NAD(P)H and ATP levels were also increased by one to two times and three to five times, respectively. Furthermore, a comparative transcriptome analysis was carried out in order to determine the mechanism involved in the enhanced activity of the Cbei_4110-inactivated mutant strain. This strategy may be extended for making industrial bio-butanol more economically attractive.

Pressure Reduction Enhancing the Production of 5-Hydroxymethylfurfural from Glucose in Aqueous Phase Catalysis System.

5-hydroxymethylfurfural (HMF) obtained from biomass is an important platform chemical for the next generation of plastics and biofuel production. Although industrialized, the high yield of HMF in aqueous systems was rarely achieved. The main problem is that HMF tends to form byproducts when co-adsorbed with water at acid sites. In this study, the pressure was reduced to improve the maximum yield of HMF from 9.3 to 35.2% (at 190 °C in 60 min) in a glucose aqueous solution. The mechanism here involved water boiling as caused by pressure reduction, which in turn promoted the desorption of HMF from the solid catalyst, thereby inhibiting the side reaction of HMF. Furthermore, the solid catalysts could be reused three times without a significant loss of their catalytic activity. Overall, this work provides an effective strategy to improve the yield of HMF in water over heterogeneous catalysts in practice.

Impact of centrifugation treatment on enzymatic hydrolysis of cellulose and xylan in poplar fibers with high lignin content.

Centrifugation is very common in the production and treatment of lignocellulose for applications like pretreatment for enzymatic hydrolysis, but it is not certain whether it affects applications of lignocellulose and almost no one realizes this problem. This study investigated the effects of centrifugation on the characteristics and enzymatic hydrolysis of poplar fibers with high lignin content. The results showed that centrifugation inhibited the enzymatic hydrolysis of fiber, but fiber characteristics and enzymatic digestibility fluctuated with increasing centrifugation time. Centrifugation for about 15 min had the least effect on fiber properties while centrifugation for 30 min had the least effect on enzymatic hydrolysis. The water retention value was closely related to the enzymatic digestibility, but the pore characteristics and crystallinity index could not reflect the enzymatic accessibility of the fiber. This article will provide useful references for the enzymatic hydrolysis of lignocellulose and other high-value applications to improve production efficiency furtherly.

Study on ultrasonic-electrochemical treatment for difficult-to-settle slime water.

The treatment of slime water not only has a direct impact on the economic benefits of the coal preparation plant, but also has an important significance for the conservation and utilization of the environment and water resources in the mining area. However, under the background of increasing mechanization level of coal mining, the proportion of fine coal is increasing, and the slime water exhibits fine, high and sticky characteristics in terms of particle size, ash, and viscosity, making it difficult for the slime water to settle. In this paper, research on the treatment of difficult-to-settle slime water by ultrasound-electrochemistry is investigated. The principle of the ultrasonic-electrochemical method for treating difficult-to-settle slime water is discussed. The effects of electrodes, electrolytes, ultrasonic energy density and ultrasonic time on treatment of difficult-to-settle slime water are analyzed. Results show that ultrasound-electrochemical pretreatment for difficult-to-settle slime water is not a simple superposition of the two treatment methods, and ultrasound can promote the electrochemical reaction. The optimal ultrasonic parameters are given: ultrasonic frequency, energy density and ultrasonic time are 29 kHz, 0.5 W/m3 and 4 min respectively. Based on the basic properties of the micro-charge on the surface of coal particles, the flocculation sedimentation can be promoted effectively by the combination of sonochemical and electrochemical methods. Finally, the research status of the new treatment technology for difficult-to-settle slime water is discussed, and the development trend is pointed out.

The influence of "thermal drying pretreatment" on enzymatic hydrolysis of cellulose and xylan in poplar fibers with high lignin content.

Thermal drying for lignocellulosic fibers is a common procedure in laboratories and factories, which isn't a pretreatment traditionally used to enhance cellulose conversion but inevitably occurs. This study investigated the effects of thermal drying conditions on the characteristics and enzymatic hydrolysis of lignocellulosic fiber granules with high lignin content. The results showed that fiber characteristics decreased linearly with the increase of temperature, which caused the linear reduction of enzymatic digestibility. Then, the increase of time caused the exponential decrease of fiber characteristics and enzymatic digestibility. Moreover, the reduction of initial water content obtained by centrifugation resulted in almost the same porosity and slightly increased water retention value (WRV) of fibers, which caused slight changes of lignocellulose digestibility. Finally, repeated drying and swelling led to complex changes in fiber characteristics, which caused fluctuations in enzymatic hydrolysis with a downward trend. This article will provide a reference for lignocellulose enzymatic hydrolysis.

The New NNRTI ACC007 Combined with Lamivudine and Tenofovir Disoproxil Fumarate Show Synergy Anti-HIV Activity In Vitro.

BACKGROUND: Acquired immunodeficiency syndrome can hardly be cured currently and people with human immunodeficiency virus (HIV) need lifelong treatment that may result in the emergence of drug resistance which leads to failed treatment. Thus, the development of new anti- HIV drugs and new treatment regimens are necessary. OBJECTIVE: The aim of this study is to analyze the combined anti-HIV activity of tenofovir disoproxil fumarate, lamivudine and ACC007, a new non-nucleoside reverse transcriptase inhibitor. METHODS: The antiviral activity of tenofovir disoproxil fumarate, lamivudine and ACC007 alone or in combination against different HIV-1 strains was determined by the detection of HIV-1 p24 level through enzyme-linked immunosorbent assay. RESULT: ACC007 showed EC50 of nanomolar range (from 3.03 nM to 252.59 nM) against all HIV-1 strains used in this study except the HIV-1A17, with EC50 of 1.57 µM. The combined antiviral activity of ACC007, lamivudine and tenofovir disoproxil fumarate showed synergy antiviral activity against all HIV-1 strains used in this study. The three-drug combination showed moderate synergism against HIV-1A17, HIV-14755-5, HIV-1K103N and HIV-1V106M, with a combination index value ranging from 0.71 to 0.87, and showed synergism against the other HIV-1 strains with combination index value from 0.35 to 0.67. The combination with ACC007 significantly increases the dose reduction index value of lamivudine and tenofovir disoproxil fumarate, compared with two-drug combination. CONCLUSION: ACC007 exhibits potent antiviral activity alone or with 3TC and TDF, and exerts synergistic effect against all HIV strains used in our investigation in vitro.

Comparative transcriptomic analysis of Clostridium acetobutylicum biofilm and planktonic cells.

Biofilm-based immobilization of solventogenic Clostridia has been extensively exploited to overcome traditional bottlenecks in biobutanol production like solvent toxicity and low productivities. However, the molecular basis of solventogenic Clostridia biofilm is rarely explored. Here, for the first time, we report DNA array-based study of Clostridium acetobutylicum biofilm cells to elucidate the transcriptional modulation. Results showed that 16.2% of the C. acetobutylicum genome genes within the biofilm cells were differentially expressed, with most genes being up-regulated. The most dramatic changes occurred with amino acid biosynthesis, with sulfur uptake and cysteine biosynthesis being the most up-regulated and histidine biosynthesis being the most down-regulated in the biofilm cells. It was demonstrated that C. acetobutylicum biofilm cells increased metabolic activities probably by up-regulating iron and sulfur uptake and Fe-S cluster biosynthesis genes as well as glycolysis genes. Furthermore, genes involved in sporulation, granulose formation, extracellular polymer degradation, pentose catabolisms, and various other processes were also notably regulated, indicating that the biofilm mode of growth rendered the cells a distinct phenotype. This study provides valuable insights into the transcriptional regulation in C. acetobutylicum biofilm cells and should be highly useful for understanding and developing the biofilm-based processes.

Enhanced production of butanol and acetoin by heterologous expression of an acetolactate decarboxylase in Clostridium acetobutylicum.

Butanol is an important industrial chemical and an attractive transportation fuel. However, the deficiency of reducing equivalents NAD(P)H in butanol fermentation results in a large quantity of oxidation products, which is a major problem limiting the atom economy and economic viability of bio-butanol processes. Here, we integrated the butanol fermentation process with a NADH-generating, acetoin biosynthesis process to improve the butanol production. By overexpressing the α-acetolactate decarboxylase gene alsD from Bacillus subtilis in Clostridium acetobutylicum, acetoin yield was significantly increased at the cost of acetone. After optimization of fermentation conditions, butanol (12.9g/L), acetoin (6.5g/L), and ethanol (1.9g/L) were generated by the recombinant strain, with acetone no more than 1.8g/L. Thus, both mass yield and product value were greatly improved. This study demonstrates that reducing power compensation is effective to improve the atom economy of butanol fermentation, and provides a novel approach to improve the economic viability of bio-butanol production.

A water-forming NADH oxidase regulates metabolism in anaerobic fermentation.

BACKGROUND: Water-forming NADH oxidase can oxidize cytosolic NADH to NAD(+), thus relieving cytosolic NADH accumulation in Saccharomyces cerevisiae. Previous studies of the enzyme were conducted under aerobic conditions, as O2 is the recognized electron acceptor of the enzyme. In order to extend its use in industrial production and to study its effect on anaerobes, the effects of overexpression of this oxidase in S. cerevisiae BY4741 and Clostridium acetobutylicum 428 (Cac-428) under anaerobic conditions were evaluated. RESULTS: Glucose was exhausted in the NADH oxidase-overexpressing S. cerevisiae strain (Sce-NOX) culture after 26 h, while 43.51 ± 2.18 g/L residual glucose was left in the control strain (Sce-CON) culture at this time point. After 30 h of fermentation, the concentration of ethanol produced by Sce-NOX reached 36.28 ± 1.81 g/L, an increase of 56.38 % as compared to Sce-CON (23.20 ± 1.16 g/L), while the byproduct glycerol was remarkably decreased in the culture of Sce-NOX. In the case of the C. acetobutylicum strain (Cac-NOX) overexpressing NADH oxidase, glucose consumption, cell growth rate, and the production of acetone-butanol-ethanol (ABE) all decreased, while the concentrations of acetic acid and butyric acid increased as compared to the control strain (Cac-CON). During fermentation of Cac-CON and Cac-NOX in 100-mL screw-capped bottles, the concentrations of ABE increased with increasing headspace. Additionally, several alternative electron acceptors in C. acetobutylicum fermentation were tested. Nitroblue tetrazolium and 2,6-dichloroindophenol were lethiferous to both Cac-CON and Cac-NOX. Methylene blue could relieve the effect caused by the overexpression of the NADH oxidase on the metabolic network of C. acetobutylicum strains, while cytochrome c aggravated the effect. CONCLUSIONS: The water-forming NADH oxidase could regulate the metabolism of both the S. cerevisiae and the C. acetobutylicum strains in anaerobic conditions. Thus, the recombinant S. cerevisiae strain might be useful in industrial production. Besides the recognized electron acceptor O2, methylene blue and/or the structural analogs may be the alternative elector acceptor of the NADH oxidase in anaerobic conditions.

Engineering Clostridium beijerinckii with the Cbei_4693 gene knockout for enhanced ferulic acid tolerance.

A mutant strain of Clostridium beijerinckii NCIMB 8052, C. beijerinckii M11, which exhibited ferulic acid tolerance up to 0.9g/L, was generated using atmospheric pressure glow discharge and high-throughput screening. Comparative genomic analysis revealed that this strain harbored a mutation of the Cbei_4693 gene, which encodes a hypothetical protein suspected to be an NADPH-dependent FMN reductase. After disrupting the Cbei_4693 gene in C. beijerinckii NCIMB 8052 using the ClosTron group II intron-based gene inactivation system, we obtained the Cbei_4693 gene inactivated mutant strain, C. beijerinckii 4693::int. Compared with C. beijerinckii NCIMB 8052, 6.23g/L of butanol was produced in P2 medium containing 0.5g/L of ferulic acid by 4693::int, and the ferulic acid tolerance was also significantly increased up to 0.8g/L. These data showed, for the first time, that the Cbei_4693 gene plays an important role in regulating ferulic acid tolerance in ABE fermentation by C. beijerinckii.

Biomechanical profiling of Caenorhabditis elegans motility.

Caenorhabditis elegans locomotion is a stereotyped behavior that is ideal for genetic analysis. We integrated video microscopy, image analysis algorithms, and fluid mechanics principles to describe the C. elegans swim gait. Quantification of body shapes and external hydrodynamics and model-based estimates of biomechanics reveal that mutants affecting similar biological processes exhibit related patterns of biomechanical differences. Therefore, biomechanical profiling could be useful for predicting the function of previously unstudied motility genes.