Engineering Clostridium saccharoperbutylacetonicum for Enhanced Isopropanol-Butanolethanol (IBE) Production from Lignocellulosic Biomass through Acetic Acid Pretreatment

The objective of this study was to achieve efficient biofuel production from lignocellulosic biomass through the development of a novel biomass pretreatment method in combination with the metabolic engineering of microbial strains for efficient conversion of biomass feedstock. First, an innovative biomass pretreatment method was developed using acetic acid (AA) as the treatment reagent considering its various advantages compared to the conventional dilute acid pretreatment method and the benefit of AA for biobutanol production; then, the hyper-butanol producing strain Clostridium saccharoperbutylacetonicum N1-4 was engineered for enhanced acid re-assimilation and acetone-butanol-ethanol (ABE) production from the acetic-acid-pretreated biomass; further, C. saccharoperbutylacetonicum was engineered for enhanced production of isopropanol-butanol-ethanol (IBE; which can be used directly as a fuel source rather than ABE) from the acetic-acid-pretreated biomass. For the biofuel production from lignocellulosic biomass, most biomass pretreatment processes need to use some chemical reagent as the catalyst to overcome the biomass recalcitrance barrier. Such reagents are usually severe inhibitors for the subsequence fermentation process. Therefore, in many cases, the liquid prehydrolysates fraction (LPF) after the pretreatment is discarded, which is a tremendous wasting of materials and leads to additional pollution. Biobutanol produced from ABE fermentation process has been of great interests recently due to its high value as a biofuel or biochemical. During the ABE fermentation, AA is produced and then re-assimilated as a carbon source. Thus, AA is a substrate rather than an inhibitor for biobutanol production. In this study, we employed AA as the chemical catalyst for the pretreatment of switchgrass which then be used for ABE production through simultaneous saccharification and fermentation (SSF) with hyper-butanol producing C. saccharoperbutylacetonicum N1-4. Through systematic investigation of pretreatment conditions and fermentation, we concluded that the pretreatment with 3 g/L AA at 170 oC for 20 min is the optimal conditions for switchgrass pretreatment leading to efficient biobutanol production. Both LPF and solid cellulosic fraction (SCF) of the pretreatment biomass are highly fermentable. In the fermentation with the LPF/SCF mixture, 8.6 g/L butanol (corresponding to a yield of 0.16 g/g) was obtained. Overall, here we demonstrated an innovative biomass pretreatment strategy for efficient carbon source utilization and biobutanol production. ABE fermentation generally has two phases: in the acidogenesis phase, fatty acids (acetic acid and butyric acid) are accumulated, while in the solventogenic phase, fatty acids are re-assimilated and converted into solvents. Therefore, the improvement of acid re-assimilation capability in the Clostridium host can possibly enhance the solvent production. In addition, acetic acid is often a significant component in the biomass prehydrolysates after pretreatment (especially when acid-based biomass pretreatment approach is employed). Thus, the enhancement of acid re-assimilation in Clostridium has practical significance for biofuel production from lignocellulosic biomass. Here, we overexpressed key genes of the ABE fermentation pathways in C. saccharoperbutylacetonicum to enhance the acid re-assimilation and solvent production. First, the native sol operon (ald-ctfA-ctfB-bcd) was overexpressed under the strong constitutive thiolase promoter (Pthl), generating PW2 strain. Fermentation results demonstrated that the acid re-assimilation was improved in the host strain and ABE production has been increased to 31.4 g/L (vs. 26.4 g/L in JZ100 strain as the control). Although the ethanol production has been increased by six times, the butanol production has not been significantly increased in the engineered strain. In order to further drive the carbon flux from C2 metabolites to C4 metabolites and ultimate butanol production, the key genes including hbd, thl, crt and bcd (expression cassette, or EC) in the butanol production pathway was further overexpressed under Pthl besides the sol operon overexpression as in PW2, generating PW3 strain. Compared to the control, the butanol and acetone production in PW3 was increased by 8% and 18% respectively. The final total solvent production increased by 12.4% than the control, but was 10% lower than PW2 (mainly because of the dramatic increase of ethanol production in PW2). In PW3, both sol operon and EC were overexpressed with Pthl, which could lead to competition for the same RNA polymerase for the expression of multiple genes. To avoid this issue and further improve ABE production, a new strain PW4 was constructed to express sol operon with Pthl but EC with ferredoxin gene promoter (Pfdx). The fermentation results demonstrated that, however, the production of all the solvents in PW4 was actually slightly lower than those in PW3. Moreover, we evaluated the effect of acetic acid concentrations on the solvent production in the engineered strains, and the maximum level of solvent production was achieved when 4.6 g/L acetate was supplemented. Therefore, SSF was carried out with PW2 and PW3 using switchgrass biomass pretreated with 3 g/L acetic acid (which ends up with approximately 4.6 g/L in the fermentation medium). 15.4 g/L total ABE (with a yield of 0.31 g/g) was produced in both PW2 and PW3, which was significantly higher than that in JZ100. This study demonstrated that the overexpression of key genes for acid re-assimilation and solvent production can significantly enhance ABE production in solventogenic clostridia. Acetone is highly corrosive to engine parts, and thus cannot be used as a fuel source. For this reason, the acetone produced during ABE fermentation is often considered as an undesirable byproduct. Biologically, acetone can be converted into isopropanol by the secondary alcohol dehydrogenase. Isopropanol, and thus the isopropanol-butanol-ethanol (IBE) mixture, can be used a valuable biofuel. In this study, we attempt to metabolically engineer the hyper-ABE producing C. saccharoperbutylacetonicum N1-4 strain for IBE production. First, we overexpressed the secondary alcohol dehydrogenase (sadh) gene from C. beijerinckii B593 in C. saccharoperbutylacetonicum on a plasmid, generating PW5 strain. A hydG gene (encoding a putative electron transfer protein) is right downstream of sadh within the same operon in the C. beijerinckii B593 genome. Therefore, additionally, we overexpressed sadh-hydG gene cluster together in C. saccharoperbutylacetonicum to evaluate the effect of hydG for isopropanol production, generating PW6 strain. Fermentation results indicated that in both PW5 and PW6, high levels of isopropanol were produced with no acetone production was detected. Comparatively, PW6 produced slightly higher isopropanol (10.2 g/L vs. 9.4 g/L in PW5) and total IBE. However, overall the performance of PW6 for solvent production is very similar to that of PW5. To eliminate the issue with plasmid-based overexpression such as instability and the requirement of antibiotics for cell cultivation and fermentation, we further integrated sadh or sadh-hydG into the chromosome of C. saccharoperbutylacetonicum, and generated strains PW8 and PW9. In PW8, there was 4.8 g/L acetone and 4.0 g/L isopropanol produced, while in PW9, up to 9.5 g/L isopropanol was produced with only 0.4 g/L acetone was detected. This indicated that the co-overexpression of hydG with sadh through chromosomal integration had significant positive effects on the conversion of acetone to isopropanol. In order to further enhance the solvent production, we additionally overexpressed in PW9 the sol operon (ald-ctfA-ctfB-adc), the expression cassette EC (thl-hbd-crt-bcd), or sol in combination with EC, generating strains PW10, PW11, and PW12, respectively. The fermentation characterization indicated that PW10 had significantly elevated ethanol production, as well as 25% higher isopropanol with slightly decreased butanol production, leading to a significant increase in total solvent titer (34.2 g/L vs. 27.6 g/L in PW9) and yield (0.48 g/g vs. 0.40 g/g in PW9). In PW11, the butanol production increased to 17.9 g/L while ethanol production decreased to 0.4 g/L; however, the isopropanol and final total solvent production was very similar to that in PW9. In PW12, with the co-overexpression of sol operon and EC, the production of isopropanol, butanol, and ethanol increased to 11.7 g/L, 17.3 g/L, and 1.1 g/L respectively comparing to PW9, resulting in a slight increase in total solvent yield. Finally, SSF was carried out with PW9 and PW10 using the acetic-acid-pretreated switchgrass as the feedstock, and the final solvent titer reached 13.7 g/L and 16.2 g/L, corresponding to the solvent yield of 0.27 g/L and 0.32 g/g in PW9 and PW10, respectively. The engineered strains in this study (PW9, PW10, PW11) produced the highest total IBE that has ever been reported in the batch fermentation with solventogenic clostridia. Our results indicated that the acetic-acid-pretreated biomass can be efficiently converted into biofuel using the metabolically engineered Clostridium hosts. Overall, this study demonstrated an innovative approach for biofuel production by combining a tailored biomass pretreatment method and metabolic engineering of microbial workhorse for enhanced conversion of lignocellulosic carbon source for biofuel production

Tribological Performance of Advanced Polymeric Coatings Under Extreme Operating Conditions

Polymers and their composites have favorable tribological performance such as low coefficient of friction (COF) and good corrosion resistance, when working as bearing materials. The present work is studying the tribological performance of thin (~ 10s of microns) high-bearing polymeric coatings under extreme working conditions, including high temperature, cryogenic temperature, high contact pressure, high chamber pressure, starved lubrication, and abrasive wear. This work is an important contribution in proving the concept of application of thin polymeric coatings in environments such as dry sliding bearing, valve sealing surfaces, hydrodynamic bearings and drilling application under different extreme working conditions. Three groups of polymers, namely Polytetrafluoroethylene (PTFE)-based, Polyether ether ketone (PEEK)-based, and Aromatic Thermosetting coPolyesters(ATSP)-based coatings were extensively studied. Out of the three groups of polymers, ATSP-based coating showed the most desirable tribological performance: ‘zero wear’ at different temperature from -160°C to 260°C with dry sliding, extremely low wear coefficient (4.15×10⁻⁸ mm³/Nm) under starved lubrication condition, stable coefficient of friction (COF) and low wear rate under sand abrasive condition, and extreme low COF for oil and gas drilling application. Traditionally, the friction force between two solids is attributed to adhesion and deformation effects; where the adhesion force involves the shearing between the real contact surfaces and deformation is due to the hard material‘s asperities plowing on the softer material. This work proposes a phenomenological model of friction for viscoelastic materials by using the viscosity and elasticity parameters acquired by nano-indentation measurements at elevated temperatures. Substituting the viscosity and elastic modulus terms, the model showed reasonable COF for the coatings up to temperatures that were lower than the glass transition temperature

Tribological Performance of Advanced Polymeric Coatings Under Extreme Operating Conditions

Polymers and their composites have favorable tribological performance such as low coefficient of friction (COF) and good corrosion resistance, when working as bearing materials. The present work is studying the tribological performance of thin (~ 10s of microns) high-bearing polymeric coatings under extreme working conditions, including high temperature, cryogenic temperature, high contact pressure, high chamber pressure, starved lubrication, and abrasive wear. This work is an important contribution in proving the concept of application of thin polymeric coatings in environments such as dry sliding bearing, valve sealing surfaces, hydrodynamic bearings and drilling application under different extreme working conditions. Three groups of polymers, namely Polytetrafluoroethylene (PTFE)-based, Polyether ether ketone (PEEK)-based, and Aromatic Thermosetting coPolyesters(ATSP)-based coatings were extensively studied. Out of the three groups of polymers, ATSP-based coating showed the most desirable tribological performance: ‘zero wear’ at different temperature from -160°C to 260°C with dry sliding, extremely low wear coefficient (4.15×10⁻⁸ mm³/Nm) under starved lubrication condition, stable coefficient of friction (COF) and low wear rate under sand abrasive condition, and extreme low COF for oil and gas drilling application. Traditionally, the friction force between two solids is attributed to adhesion and deformation effects; where the adhesion force involves the shearing between the real contact surfaces and deformation is due to the hard material‘s asperities plowing on the softer material. This work proposes a phenomenological model of friction for viscoelastic materials by using the viscosity and elasticity parameters acquired by nano-indentation measurements at elevated temperatures. Substituting the viscosity and elastic modulus terms, the model showed reasonable COF for the coatings up to temperatures that were lower than the glass transition temperature

Renewable fatty acid ester production in Clostridium.

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses

Which method is best for the induction of labour?: A systematic review, network meta-analysis and cost-effectiveness analysis

Background: More than 150,000 pregnant women in England and Wales have their labour induced each year. Multiple pharmacological, mechanical and complementary methods are available to induce labour. Objective: To assess the relative effectiveness, safety and cost-effectiveness of labour induction methods and, data permitting, effects in different clinical subgroups. Methods: We carried out a systematic review using Cochrane methods. The Cochrane Pregnancy and Childbirth Group’s Trials Register was searched (March 2014). This contains over 22,000 reports of controlled trials (published from 1923 onwards) retrieved from weekly searches of OVID MEDLINE (1966 to current); Cochrane Central Register of Controlled Trials (The Cochrane Library); EMBASE (1982 to current); Cumulative Index to Nursing and Allied Health Literature (1984 to current); ClinicalTrials.gov; the World Health Organization International Clinical Trials Registry Portal; and hand-searching of relevant conference proceedings and journals. We included randomised controlled trials examining interventions to induce labour compared with placebo, no treatment or other interventions in women eligible for third-trimester induction. We included outcomes relating to efficacy, safety and acceptability to women. In addition, for the economic analysis we searched the Database of Abstracts of Reviews of Effects, and Economic Evaluations Databases, NHS Economic Evaluation Database and the Health Technology Assessment database. We carried out a network meta-analysis (NMA) using all of the available evidence, both direct and indirect, to produce estimates of the relative effects of each treatment compared with others in a network. We developed a de novo decision tree model to estimate the cost-effectiveness of various methods. The costs included were the intervention and other hospital costs incurred (price year 2012–13). We reviewed the literature to identify preference-based utilities for the health-related outcomes in the model. We calculated incremental cost-effectiveness ratios, expected costs, utilities and net benefit. We represent uncertainty in the optimal intervention using cost-effectiveness acceptability curves. Results: We identified 1190 studies; 611 were eligible for inclusion. The interventions most likely to achieve vaginal delivery (VD) within 24 hours were intravenous oxytocin with amniotomy [posterior rank 2; 95% credible intervals (CrIs) 1 to 9] and higher-dose (≥ 50 μg) vaginal misoprostol (rank 3; 95% CrI 1 to 6). Compared with placebo, several treatments reduced the odds of caesarean section, but we observed considerable uncertainty in treatment rankings. For uterine hyperstimulation, double-balloon catheter had the highest probability of being among the best three treatments, whereas vaginal misoprostol (≥ 50 μg) was most likely to increase the odds of excessive uterine activity. For other safety outcomes there were insufficient data or there was too much uncertainty to identify which treatments performed ‘best’. Few studies collected information on women’s views. Owing to incomplete reporting of the VD within 24 hours outcome, the cost-effectiveness analysis could compare only 20 interventions. The analysis suggested that most interventions have similar utility and differ mainly in cost. With a caveat of considerable uncertainty, titrated (low-dose) misoprostol solution and buccal/sublingual misoprostol had the highest likelihood of being cost-effective. Limitations: There was considerable uncertainty in findings and there were insufficient data for some planned subgroup analyses. Conclusions: Overall, misoprostol and oxytocin with amniotomy (for women with favourable cervix) is more successful than other agents in achieving VD within 24 hours. The ranking according to safety of different methods was less clear. The cost-effectiveness analysis suggested that titrated (low-dose) oral misoprostol solution resulted in the highest utility, whereas buccal/sublingual misoprostol had the lowest cost. There was a high degree of uncertainty as to the most cost-effective intervention

Tribology of High-performance PEEK-, PI-, and ATSP-based Self-lubricating Polymers Up to 300 oC

Abstract High-performance polymers (HPPs) with self-lubricating properties are promising materials for bearing and tribological components that demand low friction and low wear in the absence of liquid lubrication. This study reports on the tribological performance of three advanced HPPs, namely ATSP-, PEEK-, and PI-based polymer composites. The experiments were performed using pin-on-disk configuration under dry sliding conditions and different environmental temperatures from 25 (room temperature) to 300 °C. The role of temperature on the formation of polymer transfer films on the steel counterpart was investigated using microscopy and profilometric measurements, and correlations were made to their tribological performance. From the three tested composites, ATSP-based composite exhibited the best overall performance with low friction and low wear.</jats:p

Tai Ji Quan: An overview of its history, health benefits, and cultural value

AbstractTai Ji Quan is considered to be a part of traditional Chinese Wushu (a martial art) and comprises various styles that have evolved historically from the Chen, Yang, Wǔ, Wú, and Sun families (schools). Recent simplification of the original classic styles has made Tai Ji Quan easier to adopt in practice. Thus, the traditional legacy of using Tai Ji Quan for self-defense, mindful nurturing of well-being, and fitness enhancement has been expanded to more contemporary applications that focus on promoting physical and mental health, enhancing general well-being, preventing chronic diseases, and being an effective clinical intervention for diverse medical conditions. As the impact of Tai Ji Quan on physical performance and health continues to grow, there is a need to better understand its historical impact and current status. This paper provides an overview of the evolution of Tai Ji Quan in China, its functional utility, and the scientific evidence of its health benefits, as well as how it has been a vehicle for enhancing cultural understanding and exchanging between East and West

Advanced polymeric coatings and their applications: green tribology

In this article, different advanced polymeric coatings and their applications are reviewed. Polymeric coatings have broad properties and functions; herein we discuss the coatings’ deposition methods and three different functional advanced polymeric coatings, namely, tribological coatings, superhydrophobic coatings and self-healing coatings. Advanced tribological polymeric coatings such as polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), and aromatic thermosetting copolyester (ATSP) exhibited excellent wear resistance and low friction, when sliding against different counter surfaces. Hydrophobic coatings prevent the coatings from water wetting by their surface structure and low surface energy, and self-healing coatings can recover or maintain their functionality after damageAbstract. Keywords. Introduction. Polymer Coating Deposition Techniques for Advanced Tribological Applications. Tribology of Polymer Coatings Under Extreme Working Conditions. Hydrophobic Coatings and Their Applications. Self-Healing Coatings and Their Applications. Conclusions. Reference