Monitoring CaCO3 Content in Recycled Polypropylene with Raman Spectrometry.

As a commonly used filler, CaCO3 frequently finds its way into recycled polypropylene (rPP) as a contaminant during the mechanical recycling process. Given the substantial impact of CaCO3 on the properties of PP materials, close monitoring of their content is important to ensure the quality of rPP. In the present work, Raman spectrometry was employed to develop a rapid, accurate, and convenient method for determining CaCO3 content in rPP. Partial least-squares (PLS) regression was used to construct prediction models. Various spectrum pretreatment methods, including multivariate scatter correction (MSC), standard normal variate transformation (SNV), smoothing, and first derivative, were investigated to improve the model performance. In independent validation, the optimal PLS model reached an R 2 of 0.9735 and a root-mean-square error of prediction (RMSEP) of 2.7786 CaCO3 wt %. Furthermore, linear and second-order polynomial regressions, utilizing the intensity ratios of characteristic CaCO3 and PP Raman peaks, were conducted. The most effective quadratic regression curve demonstrated superior independent validation performance with an R 2 of 0.9926 and an RMSEP of 1.6999 CaCO3 wt %. Validation with recycled PP samples confirmed that the quadratic regression was more accurate and reliable to quantify CaCO3 in rPP. The observed quadratic relationship between the CaCO3 and PP Raman peak intensity ratio and the CaCO3 wt % can be attributed to the significant difference in the densities of the two components. The outcomes of this research will help to facilitate the proper recycling of PP materials.

Reactive Blending of Recycled Poly(ethylene terephthalate)/Recycled Polypropylene: Kinetics Modeling of Non-Isothermal Crystallization.

Plastics were developed to change our world for the better. However, plastic pollution has become a serious global environmental crisis. Thermoplastic polyesters and polyolefins are among the most abundant plastic waste. This work presents an in-depth non-isothermal crystallization kinetics analysis of recycled post-consumer poly(ethylene terephthalate) (rPET) and recycled polypropylene (rPP) blends prepared through reactive compounding. The effect of pyromellitic dianhydride (PMDA) on crystallization kinetics and phase morphology of rPET/rPP blends was investigated by differential scanning calorimetry (DSC) and microscopy techniques. DSC results showed that increasing rPP content accelerated rPET crystallization while reducing crystallinity, which indicates the nucleation effect of the rPP phase in blends. Further, it was found that the incorporation of PMDA increased the degree of crystallinity during non-isothermal crystallization, even though the rate of crystallinity decreased slightly due to its restriction effects. The non-isothermal crystallization kinetics was analyzed based on the theoretical models developed by Jeziorny, Ozawa, Mo, and Tobin. The activation energy of the crystallization process derived from Kissinger, Takhor, and Augis-Bennett models was found to increase in rPET/rPP blends with increasing PMDA due to hindered dynamics of the system. Rheological measurements revealed that rPET melt viscosity is remarkably increased in the presence of PMDA and reactive blending with rPP relevant for processing. Moreover, nanomechanical mapping of the rPP phase dispersed in the rPET matrix demonstrated the broadening of the interfacial domains after reactive blending due to the branching effect of PMDA. Findings from this study are essential for the recycling/upcycling thermoplastics through non-isothermal fabrication processes, such as extrusion and injection molding, to mitigate the lack of sorting options.

Silver nanoparticles induced with aqueous black carpenter ant extract selectively inhibit the growth of Pseudomonas aeruginosa.

Aqueous black carpenter ant extract (ABCAE) was used to synthesize silver nanoparticles (AgNPs). The ABCAE was rich in water-soluble compounds such as hydrophilic polypeptides that behaved as both reducing and stabilizing agents for generating AgNPs from Ag+ ion precursors. The diameter of the observed AgNPs was mostly in the range of 20-60 nm. The AgNPs were tested as an antibacterial agent for the growth inhibition of two pathogenic bacteria (Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 27661) and one common bacteria (Escherichia coli K12 ATCC 10798). Disk diffusion test showed that the AgNPs selectively inhibited the growth of P. aeruginosa but not for the other two species, suggesting the potential application of the green-chemically synthesized AgNPs as a selective antibacterial agent without harming other beneficial bacteria.

Synthesis and Characterization of Quercetin-Iron Complex Nanoparticles for Overcoming Drug Resistance.

Quercetin, one of the major natural flavonoids, has demonstrated great pharmacological potential as an antioxidant and in overcoming drug resistance. However, its low aqueous solubility and poor stability limit its potential applications. Previous studies suggest that the formation of quercetin-metal complexes could increase quercetin stability and biological activity. In this paper, we systematically investigated the formation of quercetin-iron complex nanoparticles by varying the ligand-to-metal ratios with the goal of increasing the aqueous solubility and stability of quercetin. It was found that quercetin-iron complex nanoparticles could be reproducibly synthesized with several ligand-to-iron ratios at room temperature. The UV-Vis spectra of the nanoparticles indicated that nanoparticle formation greatly increased the stability and solubility of quercetin. Compared to free quercetin, the quercetin-iron complex nanoparticles exhibited enhanced antioxidant activities and elongated effects. Our preliminary cellular evaluation suggests that these nanoparticles had minimal cytotoxicity and could effectively block the efflux pump of cells, indicating their potential for cancer treatment.

Highly Efficient Genome Editing in Clostridium difficile Using the CRISPR-Cpf1 System.

Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Here, we describe a streamlined CRISPR-Cpf1-based protocol to achieve precise genome editing in C. difficile with high efficiencies. Our work highlighted the first application of CRISPR-Cpf1 for genome editing in C. difficile, which are both crucial for understanding pathogenic mechanism of C. difficile and developing strategies to fight against C. difficile infection (CDI). In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. Approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.

Sorption and recovery of phenolic compounds from aqueous phase of sewage sludge hydrothermal liquefaction using bio-char.

Bio-char, a by-product of thermochemical conversion processes, has a great potential in phenolic compounds sorption from the waste aqueous phase produced from the hydrothermal liquefaction (HTL) process while being a low-cost sorbent. This study investigated the effect of temperature, pH, bio-char concentration, and mixing speed on two types of bio-char sorption of phenolic compounds using Taguchi's design of experiment and response surface method. Isothermal kinetics and thermodynamic properties were also evaluated to explain the sorption mechanism. The experimental results were well described by the pseudo-second-order kinetic model for both types of bio-char. The Langmuir isotherm model was found to be more suitable at high sorption temperatures, while the Freundlich isotherm model was better at low temperatures. Finally, the alkaline desorption and regeneration experiments were examined, and the eluents with phenolic compounds were characterized using a liquid chromatography-mass spectrometer.

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.

Effect of ammonia removal and biochar detoxification on anaerobic digestion of aqueous phase from municipal sludge hydrothermal liquefaction.

Hydrothermal liquefaction is a promising method to convert municipal sludge into an energy-dense fuel. The inevitable by-product aqueous phase is rich in complex organics, which has the potential for energy and nutrient recovery and can be treated by anaerobic digestion to produce methane. However, toxic compounds such as ammonia and phenolics present would inhibit the function of micro-organisms. This study investigated the influence of ammonia and phenolics removal on anaerobic digestion. The results showed that the treated aqueous phase resulted in up to 225 ml CH4/g COD. The highest methane production was obtained in the culture with both ammonia and phenolics removal at pH 7.0, which was about 90% higher than only ammonia removal and seven times higher than only phenolics removal. The microbial community analysis results showed that these two treatments could increase microbial diversity and upregulate the relative abundance of methanogens.

RRNPP-type quorum-sensing systems regulate solvent formation, sporulation and cell motility in Clostridium saccharoperbutylacetonicum.

BACKGROUND: Clostridium saccharoperbutylacetonicum N1-4 (HMT) is a strictly anaerobic, spore-forming Gram-positive bacterium capable of hyper-butanol production through the well-known acetone-butanol-ethanol fermentation process. Recently, five putative RRNPP-type QSSs (here designated as QSS1 to QSS5) were predicted in this bacterial strain, each of which comprises a putative RRNPP-type regulator (QssR1 to QssR5) and a cognate signaling peptide precursor (QssP1 to QssP5). In addition, both proteins are encoded by the same operon. The functions of these multiple RRNPP-type QSSs are unknown. RESULTS: To elucidate the function of multiple RRNPP-type QSSs as related to cell metabolism and solvent production in N1-4 (HMT), we constructed qssR-deficient mutants ΔR1, ΔR2, ΔR3 and ΔR5 through gene deletion using CRISPR-Cas9 and N1-4-dcas9-R4 (with the QssR4 expression suppressed using CRISPR-dCas9). We also constructed complementation strains by overexpressing the corresponding regulator gene. Based on systematic characterization, results indicate that QSS1, QSS2, QSS3, and QSS5 positively regulate the sol operon expression and thus solvent production, but they likely negatively regulate cell motility. Consequently, QSS4 might not directly regulate solvent production, but positively affect cell migration. In addition, QSS3 and QSS5 appear to positively regulate sporulation efficiency. CONCLUSIONS: Our study provides the first insights into the roles of multiple RRNPP-type QSSs of C. saccharoperbutylacetonicum for the regulation of solvent production, cell motility, and sporulation. Results of this study expand our knowledge of how multiple paralogous QSSs are involved in the regulation of essential bacterial metabolism pathways.

Enhancement of biogas production from wastewater sludge via anaerobic digestion assisted with biochar amendment.

Studies have shown that biochar enhances methane formation due to the presence of redox active moieties and its conductive properties. This study investigated the influence of biochar, which was produced from Douglas fir pyrolysis, on biogas production and microbial community during anaerobic digestion (AD) of wastewater sludge. The results showed that biochar significantly enhances methane (CH4) production rate and increases its final yield during AD. The cumulative highest CH4 production obtaining in cultures with DF500 (biochar from Douglas fir at 500 °C) were about 11% and 98% more than the culture without biochar at 37 °C and 25 °C AD temperature, respectively. At 55 °C, the maximum CH4 yield reached 172.3 ml/g COD with DF730, which was about 48.3% more than control culture. The microbial community analysis results showed that biochar could up-regulate the role of micro-ecology especially the methanogens and improve the AD process.

Enhancement of acid re-assimilation and biosolvent production in Clostridium saccharoperbutylacetonicum through metabolic engineering for efficient biofuel production from lignocellulosic biomass.

In the clostridial acetone-butanol-ethanol (ABE) fermentation, the intermediate acetate and butyrate are re-assimilated for solvent production. Here, key genes in ABE pathways in Clostridium saccharoperbutylacetonicum N1-4 were overexpressed to enhance acid re-assimilation and solvent production. With the overexpression of sol operon, acid re-assimilation was enhanced, and ABE production was increased by 20%, with ethanol production increased by six times but almost no increase in butanol production. To further drive carbon flux for C4 metabolites and ultimate butanol production, key genes including hbd, thl, crt and bcd in butanol production pathway were further overexpressed. Compared to the control, butanol, acetone and total ABE production in the new strain was increased by 8%, 18%, and 12.4%, respectively. Finally, simultaneous saccharification and fermentation was carried out using acetate-pretreated switchgrass. 15.4 g/L total ABE (with a yield of 0.31 g/g) was produced in both engineered strains, which was significantly higher than the control.

Bacteria Co-colonizing with Clostridioides Difficile in Two Asymptomatic Patients.

BACKGROUND: Clostridium difficile infection (CDI) is the leading cause of nosocomial diarrhea. Co-colonization of key bacterial taxa may prevent the transition from asymptomatic C. difficile colonization to CDI. However, little is known about the composition of key bacterial taxa in asymptomatic patients. METHODS: In the present study, the culture method was used to examine the composition of stool microbiota in two asymptomatic patients from Guizhou, China. RESULTS: A total of 111 strains were isolated and phylogenetic relationships were determined by 16S ribosomal gene sequencing and Molecular Evolutionary Genetics Analysis version 7. The results demonstrated that Escherichia (33.3%, 37/111), Clostridium (24.3%, 27/111) and Enterococcus (11.7%, 13/111) exhibited a high ratio in asymptomatic patients. These isolates derived from two phyla: Firmicutes (51.3%, 57/111) and Proteobacteria (44.1%, 49/111). In addition, co-colonization of human pathogens Fusobacterium nucleatum, Ralstonia pickettii, Klebsiella pneumoniae, Klebsiella quasipneumoniae and Clostridium tertium with C. difficile was identified. To the best of our knowledge, these pathogens have not been co-isolated with C. difficile previously. CONCLUSIONS: In summary, the present study identified the composition of fecal microbiota in two asymptomatic patients in Guizhou, China. These results suggested that co-infection with human pathogens may be ubiquitous during CDI progression.

Co-infection of Clostridioides (Clostridium) difficile GMU1 and Bacillus cereus GMU2 in one patient in Guizhou, China.

Clostridioides (Clostridium) difficile and Bacillus cereus infections are frequently reported in human individually. However, co-infection of both pathogens in human is extremely rare. In the present study, we reported a case of human enteric disease caused by co-infection of C. difficile and B. cereus in Guizhou, China. The 16S rDNA sequencing result showed that C. difficile GMU1 and B. cereus GMU2 were most related to C. difficile ATCC 9689 and B. cereus ATCC 14579. The toxin genotype of C. difficile GMU1 and B. cereus GMU2 were tcdA+tcdB+tcdC+ and bceT+nheA+nheB+nheC+, respectively. Cytotoxicity assay demonstrated that C. difficile GMU1 produced significantly higher toxin B compare to C. difficile 630 stain. In contrast, B. cereus GMU2 has comparable NheA toxin productivity compare to previous report. The antimicrobial susceptibility test showed that the combination of ampicillin and vancomycin was most efficient to inhibit both C. difficile GMU1 and B. cereus GMU2.

Enhancement of sucrose metabolism in Clostridium saccharoperbutylacetonicum N1-4 through metabolic engineering for improved acetone-butanol-ethanol (ABE) fermentation.

This work investigated sucrose metabolism in C. saccharoperbutylacetonicum. Inactivation of sucrose catabolism operon resulted in 28.9% decrease in sucrose consumption and 44.1% decrease in ABE production with sucrose as sole carbon source. Interestingly, a large amount of colloid-like polysaccharides were generated in the mutant, which might be due to inefficient intracellular sucrose metabolism. Deletion of transcriptional repressor gene successfully alleviated CCR and enhanced ABE production by 24.7%. Additional overexpression of endogenous sucrose pathway further elevated sucrose consumption and enhanced ABE production by 17.2%, 45.7%, or 22.5% compared to wild type with sucrose, mixed sugars or sugarcane juice as substrate, respectively. The engineered strain could be a robust platform for efficient biofuel production from inexpensive sucrose-based carbon sources.

Markerless genome editing in Clostridium beijerinckii using the CRISPR-Cpf1 system.

CRISPR-Cpf1 is a type V CRISPR system that has recently been exploited for genome engineering purposes. Compared to the well-known Streptococcus pyogenes CRISPR-Cas9 system, the effector protein Cpf1 recognizes T-rich protospacer-adjacent motif (PAM) instead of G-rich PAM (used by CRISPR-Cas9), which could offer a substantial expansion of the existing genetic toolbox for genome editing. In this study, we report the implementation of the Acidaminococcus sp. Cpf1 (AsCpf1) for markerless genome engineering in Clostridium beijerinckii, a prominent species for biosolvent production through the well-known Acetone-Butanol-Ethanol (ABE) pathway. A lactose inducible promoter was used to control the expression of AsCpf1 to decrease its toxicity, while a constitutive small RNA promoter was employed to drive the expression of pre-crRNA. A One-Step-Assembly (OSA) approach was employed to construct the CRISPR-Cpf1-based vector in one single step, which simplified and streamlined the plasmid construction process. Using the customized CRISPR-Cpf1 system, we successfully deleted spo0A and pta genes in C. beijerinckii, with an editing efficiency of up to 100%. Altogether, our results demonstrated the easy programmability and high efficiency of the CRISPR-Cpf1 system for versatile genome engineering purposes. This study provides valuable guidance and essential references for repurposing the CRISPR-Cpf1 system for genome engineering in other microorganisms.

Genome Editing in Clostridium saccharoperbutylacetonicum N1-4 with the CRISPR-Cas9 System.

Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (CRISPR-Cas9) system. First, the functionality of the CRISPR-Cas9 system previously customized for Clostridium beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting pta and buk, two essential genes for acetate and butyrate production, respectively. pta and buk single and double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is <20%). Therefore, the efficiency was further optimized by evaluating various promoters for guide RNA (gRNA) expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways; however, neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield, and selectivity were improved in mutants, depending on the fermentation medium. In the pta buk double deletion mutant, the butanol production in P2 medium reached 19.0 g/liter, which is one of the highest levels ever reported from batch fermentations.IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains with desirable butanol-producing features. This tool can be easily adapted for use in closely related microorganisms. As also reported by others, here we demonstrated with solid data that the highly efficient expression of gRNA is the key factor determining the efficiency of CRISPR-Cas9 for genome editing. The protocol developed in this study can provide essential references for other researchers who work in the areas of metabolic engineering and synthetic biology. The developed mutants can be used as excellent starting strains for development of more robust ones for desirable solvent production.

[Cloning and expression of key genes of butanol synthetic pathway in Escherichia coli].

OBJECTIVE: We constructed a recombinant Escherichia coli strain for butanol production by cloning the cDNA sequence of the key butanol synthetic pathway genes from Clostridium acetobutylicum ATCC824. METHODS: We amplified the genes of thil, adhE2 and BCS operon by PCR with C. acetobutylicum ATCC824 genome as a template. We constructed the recombinant strain E. coli pBAT (BCS operon-adhE2-thil/pTrc99a/MG1655). We used 0.1 mmol/l Isopropyl beta-D-thiogalactopyranoside (IPTG) to induce the recombinant E. coli pBAT for 5 h for recombinant protein expression. We measured acetyl-CoA acetyltransferase (THL), beta-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD) and butyraldehyde dehydrogenase (BYDH)/butanol dehydrogenase (BDH) activities in E. coli MG1655 and E. coli pBAT. The fermentation of E. coli pBAT was done in flask in aerobic, micro-aerobic and anaerobic mode separately. RESULTS: In the recombinant E. coli pBAT, THL activity was 0.160 U/mg protein, about 30 times higher than that of E. coli MG1655. HBD activity was 5 times higher than that of E. coli MG1655. CRT activity was 1.53 U/mg protein whereas not detectable in E. coli MG1655. BCD activity was about 32 times higher than that of E. coli MG1655. In addition, the results show that n-butanol could be produced under anaerobic and micro-aerobic conditions. The maximum n-buntanol concentration of 84 mg/l was detected in cultivation broth. CONCLUSION: The key genes of butanol synthetic pathway were expressed in E. coli and the recombinant strains would offer an alternative strategy for butanol biosynthesis.