In Situ and Real-Time Studies, via Synchrotron X-ray Scattering, of the Orientational Order of Cellulose Nanocrystals during Solution Shearing.

In this manuscript, we report on the ordering of the cellulose nanocrystals (CNCs) as they experience shear forces during the casting process. To achieve these measurements, in situ and in real time, we used synchrotron-based grazing incidence wide-angle X-ray scattering (GIWAX). We believe that the GIWAX technique, although not commonly used to probe these types of phenomena, can open new avenues to gain deeper insights into film formation processes and surface-driven phenomena. In particular, we investigated the influence of solution concentration, shear-cast velocity, and drying temperature on the ordering of cellulose nanocrystals (CNCs) using GIWAXS. The films were prepared from aqueous suspensions of cellulose nanocrystals at two concentration values (7 and 9 wt %). As the films were cast, the X-ray beam was focused on a fixed position and GIWAXS patterns were recorded at regular time intervals. Structural characterization of the dry films was carried out via polarized optical microscopy and scanning electron microscopy. In addition, a rheological study of the CNC suspensions was performed. Our results show that the morphology of the CNC films was significantly influenced by shear velocity, concentration of the precursor suspension, and evaporation temperature. In contrast, we observed that the orientation parameter of the films was not significantly affected. The scattering intensity of the peak (200) was analyzed as a function of time, following a sigmoidal profile, hence indicating short- and long-range interactions within the anisotropic domains as they reached their final orientation state. A model capable of describing the resulting film morphologies is also proposed. The results and analysis presented in this manuscript provide new insights into the controlled alignment of cellulose nanocrystals under shear. This controlled alignment has significant implications in the development of advanced coatings and films currently used in a myriad of applications, such as catalysis, optics, electronics, and biomedicine.

Are Liquid Metals Bulk Conductors?

Stretchable electronics have potential in wide-reaching applications including wearables, personal health monitoring, and soft robotics. Many recent advances in stretchable electronics leverage liquid metals, particularly eutectic gallium-indium (EGaIn). A variety of EGaIn electromechanical behaviors have been reported, ranging from bulk conductor responses to effectively strain-insensitive responses. However, numerous measurement techniques have been used throughout the literature, making it difficult to directly compare the various proposed formulations. Here, the electromechanical responses of EGaIn found in the literature is reviewed and pure EGaIn is investigated using three electrical resistance measurement techniques: four point probe, two point probe, and Wheatstone bridge. The results indicate substantial differences in measured electromechanical behavior between the three methods, which can largely be accounted for by correcting for a fixed offset corresponding to the resistances of various parts of the measurement circuits. Yet, even accounting for several of these sources of experimental error, the average relative change in resistance of EGaIn is found to be lower than that predicted by the commonly used bulk conductor assumption, referred to as Pouillet's law. Building upon recent theories proposed in the literature, possible explanations for the discrepancies are discussed. Finally, suggestions are provided on experimental design to enable reproducible and interpretable research.

Soft Actuators Made of Discrete Grains.

Recent work has demonstrated the potential of actuators consisting of bulk elastomers with phase-changing inclusions for generating high forces and large volumetric expansions. Simultaneously, granular assemblies have been shown to enable tunable properties via different packings, dynamic moduli via jamming, and compatibility with various printing methods via suspension in carrier fluids. Herein, granular actuators are introduced, which represent a new class of soft actuators made of discrete grains. The soft grains consist of a hyperelastic shell and multiple solvent cores. Upon heating, the encapsulated solvent cores undergo liquid-to-gas phase change, inducing rapid and strong volumetric expansion of the hyperelastic shell up to 700%. The grains can be used independently for micro-actuation, or in granular agglomerates for meso- and macroscale actuation, demonstrating the scalability of the granular actuators. Furthermore, the active grains can be suspended in a carrier resin or solvent to enable printable soft actuators via established granular material processing techniques. By combining the advantages of phase-change soft actuation and granularity, this work presents the opportunity to realize soft actuators with tunable bulk properties, compatibility with self-assembly techniques, and on-demand reconfigurability.

Engineering a heterologously expressed fructosyltransferase from Aspergillus oryzae N74 in Komagataella phaffii (Pichia pastoris) for kestose production.

Fructo-oligosaccharides (FOS) are one of the most well-studied and commercialized prebiotics. FOS can be obtained either by controlled hydrolysis of inulin or by sucrose transfructosylation. FOS produced from sucrose are typically classified as short-chain FOS (scFOS), of which the best known are 1-kestotriose (GF2), 1,1-kestotetraose (GF3), and 1,1,1-kestopentaose (GF4), produced by fructosyltransferases (FTases) or ß-fructofuranosidases. In previous work, FOS production was studied using the Aspergillus oryzae N74 strain, its ftase gene was heterologously expressed in Komagataella phaffii (Pichia pastoris), and the enzyme's tertiary structure modeled. More recently, residues that may be involved in protein-substrate interactions were predicted. In this study, the aim was to experimentally validate previous in silico results by independently producing recombinant wild-type A. oryzae N74 FTase and three single-point mutations in Komagataella phaffii (Pichia pastoris). The R163A mutation virtually abolished the transfructosylating activity, indicating a requirement for the positively charged arginine residue in the catalytic domain D. In contrast, transfructosylating activity was improved by introducing the mutations V242E or F254H, with V242E resulting in higher production of GF2 without affecting that of GF3. Interestingly, initial sucrose concentration, reaction temperature and the presence of metal cofactors did not affect the enhanced activity of mutant V242E. Overall, these results shed light on the mechanism of transfructosylation of the FTase from A. oryzae and expand considerations regarding the design of biotechnological processes for specific FOS production.

Removal of E. coli and Salmonella in pot ceramic filters operating at different filtration rates.

The use of pot ceramic filters PCF to improve the domestic water quality supply has been recognized as an alternative in regions where there is unsecure water treatment or contamination of the treated water during transport and storage and an absence of safe drinking water. The aim of this study was to evaluate a model of PCF impregnated with colloidal silver under three filtration rates (1.0, 2.0 and 3.0 L/h) and three E. coli and Salmonella spp concentrations (104, 103 and 102 CFU/mL). The evaluation was made using spiked water having a turbidity of 29.9 ±â€¯4.4 NTU and conductivity of 176 ±â€¯31.7 µS/cm. The results showed a turbidity removal efficiency of 97% and average effluent of 0.9 NTU. The microbiological efficiency removal was of 2 Log Reduction Value (LRV) for E. coli and 1 LRV for Salmonella spp. There were not found significant statistical differences between the filtration rates and the removal efficiencies for turbidity E. coli and Salmonella spp. It was observed that the microbiological removal efficiency was affected by biofilm formation a phenomenon that was attributed to the presence of Salmonella spp. The combination of chemical and mechanical cleaning methods contributed to the elimination of the biofilm.

Oriented Growth of α-MnO2 Nanorods Using Natural Extracts from Grape Stems and Apple Peels.

We report on the synthesis of alpha manganese dioxide (α-MnO2) nanorods using natural extracts from Vitis vinifera grape stems and Malus domestica 'Cortland' apple peels. We used a two-step method to produce highly crystalline α-MnO2 nanorods: (1) reduction of KMnO4 in the presence of natural extracts to initiate the nucleation process; and (2) a thermal treatment to enable further solid-state growth of the nuclei. Transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM) images provided direct evidence of the morphology of the nanorods and these images were used to propose nucleation and growth mechanisms. We found that the α-MnO2 nanorods synthesized using natural extracts exhibit structural and magnetic properties similar to those of nanoparticles synthesized via traditional chemical routes. Furthermore, Fourier transform infrared (FTIR) shows that the particle growth of the α-MnO2 nanorods appears to be controlled by the presence of natural capping agents during the thermal treatment. We also evaluated the catalytic activity of the nanorods in the degradation of aqueous solutions of indigo carmine dye, highlighting the potential use of these materials to clean dye-polluted water.

Nanowire-functionalized cotton textiles.

We show the general functionalization of cotton fabrics using solution-synthesized CdSe and CdTe nanowires (NWs). Conformal coatings onto individual cotton fibers have been achieved through various physical and chemical approaches. Some involve the electrostatic attraction of NWs to cotton charged positively with a Van de Graaff generator or via 2,3-epoxypropyltrimethylammonium chloride treatments. Resulting NW-functionalized textiles consist of dense, conformal coatings and have been characterized for their UV-visible absorption as well as Raman activity. We demonstrate potential uses of these functionalized textiles through two proof-of-concept applications. The first entails barcoding cotton using the unique Raman signature of the NWs. We also demonstrate the surface-enhancement of their Raman signatures using codeposited Au. A second demonstration takes advantage of the photoconductive nature of semiconductor NWs to create cotton-based photodetectors. Apart from these illustrations, NW-functionalized cotton textiles may possess other uses in the realm of medical, anticounterfeiting, and photocatalytic applications.

A Na+:H+ antiporter and a molybdate transporter are essential for arsenite oxidation in Agrobacterium tumefaciens.

Transposon Tn5-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an Agrobacterium tumefaciens soil isolate, strain 5A. In one mutant, the transposon interrupted modB, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in mrpB, which is part of a seven-gene operon encoding an Mrp-type Na+:H+ antiporter complex. Complementation experiments with mod and mrp operons PCR cloned from the genome-sequenced A. tumefaciens strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of A. tumefaciens. As expected, the mrp mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in A. tumefaciens is involved in Na+ circulation across the membrane. Gene expression studies (lacZ reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the mrp operon in response to As(III) exposure, whereas expression of the mod operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.

Complex regulation of arsenite oxidation in Agrobacterium tumefaciens.

Seminal regulatory controls of microbial arsenite [As(III)] oxidation are described in this study. Transposon mutagenesis of Agrobacterium tumefaciens identified genes essential for As(III) oxidation, including those coding for a two-component signal transduction pair. The transposon interrupted a response regulator gene (referred to as aoxR), which encodes an ntrC-like protein and is immediately downstream of a gene (aoxS) encoding a protein with primary structural features found in sensor histidine kinases. The structural genes for As(III) oxidase (aoxAB), a c-type cytochrome (cytc2and molybdopterin biosynthesis (chlE) were downstream of aoxR. The mutant could not be complemented by aoxSR in trans but was complemented by a clone containing aoxS-aoxR-aoxA-aoxB-cytc2 and consistent with reverse transcriptase (RT) PCR experiments, which demonstrated these genes are cotranscribed as an operon. Expression of aoxAB was monitored by RT-PCR and found to be up-regulated by the addition of As(III) to cell cultures. Expression of aoxAB was also controlled in a fashion consistent with quorum sensing in that (i) expression of aoxAB was absent in As(III)-unexposed early-log-phase cells but was observed in As(III)-unexposed, late-log-phase cells and (ii) treating As(III)-unexposed, early-log-phase cells with ethyl acetate extracts of As(III)-unexposed, late-log-phase culture supernatants also resulted in aoxAB induction. Under inducing conditions, aoxS expression was readily observed in the wild-type strain but significantly reduced in the mutant, indicating that AoxR is autoregulatory and at least partially controls the expression of the aox operon. In summary, regulation of A. tumefaciens As(III) oxidation is complex, apparently being controlled by As(III) exposure, a two-component signal transduction system, and quorum sensing.

Poly(A) polymerase modification and reverse transcriptase PCR amplification of environmental RNA.

We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the alpha-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.

Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil.

Microbial populations responsible for the oxidation and reduction of As were examined in unsaturated (aerobic) soil columns treated with 75 microM arsenite [As(III)] or 250 microM arsenate [As(V)]. Arsenite [As(III)] was rapidly oxidized to As(V) via microbial activity, whereas no apparent reduction of As(V) was observed in the column experiments. Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from the same columns. Three isolates, identified as Agrobacterium tumefaciens-, Pseudomonas fluorescens-, and Variovorax paradoxus-like organisms (based on 16S sequence), were As(III) oxidizers, and all were detected in community DNA fingerprints generated by PCR coupled with denaturing gradient gel electrophoresis. The five other isolates were identified (16S gene sequence) as A. tumefaciens, Flavobacterium sp., Microbacterium sp., and two Arthrobacter sp. -like organisms and were shown to rapidly reduce As(V) under aerobic conditions. Although the two A. tumefaciens-like isolates exhibited opposite As redox activity,their 16S rDNA sequences (approximately 1400 bp) were 100% identical, and both were shown to contain putative arsC genes. Our results support the hypothesis that bacteria capable of either oxidizing As(III) or reducing As(V) coexist and are ubiquitous in soil environments, suggesting that the relative abundance and metabolic activity of specific microbial populations plays an important role in the speciation of inorganic As in soil pore waters.

Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.

A novel bacterium was cultivated from an extreme thermal soil in Yellowstone National Park, Wyoming, USA, that at the time of sampling had a pH of 3.9 and a temperature range of 65-92 degrees C. This organism was found to be an obligate aerobic, non-spore-forming rod, and formed pink-colored colonies. Phylogenetic analysis of the 16S rRNA gene sequence placed this organism in a clade composed entirely of environmental clones most closely related to the phyla Chloroflexi and Thermomicrobia. This bacterium stained gram-positive, contained a novel fatty-acid profile, had cell wall muramic acid content similar to that of Bacillus subtilis (significantly greater than Escherichia coli), and failed to display a lipopolysaccharide profile in SDS-polyacrylamide gels that would be indicative of a gram-negative cell wall structure. Ultrastructure examinations with transmission electron microscopy showed a thick cell wall (approximately 34 nm wide) external to a cytoplasmic membrane. The organism was not motile under the culture conditions used, and electron microscopic examination showed no evidence of flagella. Genomic G+C content was 56.4 mol%, and growth was optimal at 67 degrees C and at a pH of 7.0. This organism was able to grow heterotrophically on various carbon compounds, would use only oxygen as an electron acceptor, and its growth was not affected by light. A new species of a novel genus is proposed, with YNP1(T) (T=type strain) being Thermobaculum terrenum gen. nov., sp. nov. (16S rDNA gene GenBank accession AF391972). This bacterium has been deposited in the American Type Culture Collection (ATCC BAA-798) and the University of Oregon Culture Collection of Microorganisms from Extreme Environments (CCMEE 7001).