Bacterial Microarrays by Microcontact Printing: Development of a Method for Immobilizing Live Bacteria on Microarrays

Traditional microbiological experimental methods generally reveal population-wide statistics and are not capable of revealing variations between individual cells. In an effort to address this, a cheap, quick and easy means of producing micro-arrays with live bacteria immobilized on the array's coordinates has been developed. To achieve this, microcontact printing (µCP) was used to print circular PD ``islands'' onto a surface coated with either PEG or PVA. The bacteria, Pseudomonas putida KT2440 were successfully immobilized on approximately 97% of the printed islands, depending on island diameter. A LIVE/DEAD® BacLight™ assay revealed that over 99% of the immobilized bacteria survive the immobilization on the array. Qualitative analysis suggests the bacteria survive for hours after immobilization, provided nutrients are available. The developed method may, by revising the stamp design and/or choice of elastomer, likely be optimized to immobilize only one bacterium per array coordinate

Cellulose Nanofibrils as Paper Additive and Coating Material: Properties, Distribution and Interaction Effects

Cellulose is well-known as the most common bio-polymer on earth. In the cell wall of most plants, it is organized in nanoscopic structures with interesting properties for a wide range of applications, among them as an additive to paper and board - or as an applied coating. One socioeconomic perspective on these applications of the nanomaterial is associated environmental benefits; cellulose nanofibrils (CNF) as an additive to paper and board can improve tensile properties, enabling not just higher filler levels, but also increased potential fraction of recycled fibers in the product. As a coating, given its excellent oxygen barrier properties, CNF also has potential as a replacement for plastics in certain food packaging applications or, conceivably, aluminum in liquid packaging board. In the current work, we have addressed several questions pertaining to these potential applications and some fundamental questions of interest regarding the properties of CNF as individual fibrils and as fibril aggregates. Central questions in this current work, and brief answers later expounded upon, are; • Do fibril properties like crystallinity affect CNF film ductility? – Reducing crystallinity index (CI) is shown to correlate with an increased yield point and decreased Young’s modulus. • How does CNF behave within the paper or board matrix? – CNF appears to coat the surface of the furnish fraction with which it was added or premixed. – When filler particles are encased in CNF, it appears to form hollow capsules encasing the particles, and not - as one would expect - adhere very tightly. • Can CNF be successfully traced and studied within a paper matrix? – Using high resolution FIB/SEM, this has been demonstrated. – Attained resolution: 13×16×15 nm using back-scattered electron (BSE) and 7×9×15 nm for secondary electron (SE). • Are there benefits associated with premixing CNF with different furnish components? – Some benefit can be seen from premixing CNF with the filler fraction. – The effect depends on filler levels and weight fraction of added CNF. • Can CNF be applied as a barrier coating in a roll-to-roll scenario? – Highly fibrillated CNF, oxidized or not, can be used in a roll-to-roll coating situation. – Rheology plays a role in quality of applied coatings. – The mode of adhesion to the base board appears to be exclusively Physico-chemical, not mechanical. The above list gives an overview, a somewhat more expansive summary follows. Fundamentals: Crystallinity. By treatment with liquid anhydrous ammonia CI of cotton linter was reduced prior to homogenization and fibrillation. Films cast from the resulting CNF suspensions were shown to have a lower Young’s modulus and higher yield point, although crystallinity did not appear to have a significant effect on strain at break. Films of CNF treated with liquid ammonia in the same way shrank, cockled, lost mass and showed a strong increase in strain at break in addition to reduced Young’s modulus and increased yield point. Films treated with ammonia saw very large changes in mechanical properties. We conclude this is due to the cockling and shrinkage of the film itself, and not due to changes in crystallinity. Fundamentals: CNF in a paper matrix. FIB/SEM dual beam system was employed to perform investigations of CNF distribution within the paper matrix. We found the attainable resolution appears adequate to qualitatively trace CNF in volumes as large as 103 μm3 using SE. While BSE delivers reduced contrast and resolution, reduced artefacts due to surface unevenness and other effects made quantitative analysis more easily achieved using this higher-energy signal. Achieved resolutions were approximately 13×16×15 nm using BSE and 7×9×15 nm for SE. Fundamentals: Swelling of individual fibrils. Using atomic force microscopy (AFM) in liquid media (de-ionized water) and air individual fibrils were imaged in air and, later, water. As the same area was imaged the swelling of individual fibrils could be studied in detail. After 30 minute submersion, a mean swelling of 48.9 % was observed, with a standard deviation of 27.8 %. CNF-Capsule Formation. Super-resolution investigation of paper with added CNF shows the CNF appearing to form hollow capsules around filler particles. We hypothesize this is due in part to non-isometric drying. We believe it is conceivable that the surface of these capsules is drying faster, having a lower moisture content at any given time than its interior, leading to a force balance during drying favoring separation over tight conformation to the encased particle. Further investigation into the formation of such complexes by attempting to reproduce the phenomenon was begun in 2017 and continues into 2018. CNF-Premixing Strategies: Effects. We premixed mechanically produced CNF and retention aid with the long fiber fraction (LFF), the filler fraction or added them straight to (otherwise complete) furnish. We found that for high filler levels and high CNF addition levels there is a beneficial effect to dewatering times and tensile strength. Air permeability was affected similarly to dewatering times. Optical properties were not found to differ between the chosen strategies. Scanning electron microscopy (SEM) investigations of paper surfaces appears to suggest that filler particles adhere to the surface of the fraction to which CNF and retention aids were premixed. CNF as a barrier coating: Roll-to-roll application. Three qualities of CNF, TEMPO-oxidized CNF (CNF-T), carboxymethylated CNF (CNF-C) and mechanically produced CNF (CNF-M), all with and without added carboxymethylcellulose (CMC), were applied as a coating to a rough, recycled quality board. Using a single applied coating, an even, smooth surface was achieved with air resistance correlated with CNF quality, coating thickness and CMC addition. We found that lower fibril diameter correlates with improved coating. For the CNF qualities with lowest mean fibril diameter addition of CMC reduced viscosity, and appears to have created a smoother coat. We showed that SEM can be used to quantitatively investigate coating quality and gain insight into the foundation for observed air resistance values

Swelling of individual cellulose nanofibrils in water, role of crystallinity: an AFM study

Atomic force microscopy (AFM) can be used to quantitatively study nanomaterials in different media, e.g. vacuum, air, or submerged in a liquid. A technique was developed to study swelling of individual cellulose nanofibrils (CNFs) using AFM. As a case study, CNFs with different degrees of crystallinity (DoC) were examined for swellability going from dry to wet (submerged in de-ionized water). Swelling was found to depend on DoC, but no significant correlation between fibril diameter and swellability was seen. Upon introduction of de-ionized water high DoC samples (65±2%) were found to have a diameter increase of 34% on average, whereas low DoC (44±2%) were found to have a diameter increase of 44% on average. A tested control, consisting of platinum nanowires on silisium, did not swell

Mixing of cellulose nanofibrils and individual furnish components: Effects on paper properties and structure

Thermo-mechanical pulp (TMP) handsheets with different fractions of cellulose nano fibrils (CNF) and ground calcium carbonate (GCC) were made. CNF and retention chemicals were added in three different ways; to GCC, to long fibre fraction (LFF) or to complete furnish. The different addition strategies affected dewatering time, tensile strength and permeability, however opacity was not affected. Depending on filler and CNF levels, adding CNF to GCC produced the most beneficial effects on paper properties; CNF had a lower impact on dewatering times and permeability and GCC reduced strength less than for competing strategies. Adding CNF to LFF produced the least beneficial results using the same metrics. Scanning electron microscopy (SEM) analysis of the sheets reveal that sheets produced using the different strategies are structurally different; adding CNF and retention chemicals to GCC appears to have increased GCC clustering, whereas adding CNF and retention chemicals to LFF appears to have increased the fraction of GCC adsorbed on the fiber walls. CNF and retention chemical addition to complete furnish showed GCC clustering and adhering to the fiber walls, of which clustering appeared the most common

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis

Coatability of cellulose nanofibril suspensions : Role of rheology and water retention

Cellulose nanofibril (CNF) suspensions are not easily coatable because of their excessively high viscosity and yield stress, even at low solids concentrations. In addition, CNF suspensions vary widely in their properties depending on the production process used, which can affect their processability. This work reports roll-to-roll coating of three different types of CNF suspensions with a slot-die, and the influence of rheology and water retention on coatability is addressed. The impact of CMC addition on the high and low shear rate rheology, water retention, coatability, and final coating quality of these suspensions is reported. All three CNF suspensions were coated successfully using the slot-die coating process. CMC addition further improved the coatability by positively influencing both the low and high shear rate viscosity and water retention of the CNF suspensions. All CNF coatings significantly improved the air, heptane vapor, grease and oil barrier, while reducing the water vapor transmission rate to some extent.cited By 0</p

A Brief Review of Nanocellulose Based Hybrid Membranes for CO2 Separation

Due to the high specific surface area, high mechanical strength and broad possibility of surface modification, nanocellulose has obtained much attention as a new class of bio-based nanomaterials with promising potential in a wide variety of applications. Recently, a considerable amount of research has been aimed to the fabrication of nanocellulose based hybrid membranes for water treatment. However, nanocellulose based hybrid gas separation membrane is still a new research area. Herein, we force on recent advancements in the fabrication methods and separation performances of nanocellulose-based hybrid membranes for CO2 separation, the transport mechanisms involved, along with the challenges in the utilization of nanocellulose in membranes. Finally, some perspectives on future R&D of nanocellulose-based membranes for CO2 separation are proposed

The influence of temperature on cellulose swelling at constant water density

Abstract We have in this paper investigated how water sorbs to cellulose. We found that both cellulose nanofibril (CNF) and cellulose nanocrystal (CNC) films swell similarly, as they are both mainly composed of cellulose. CNF/CNC films subjected to water at 0.018 kg/m3 at 25 °C and 39 °C, showed a decrease in swelling from ~ 8 to 2%. This deswelling increased the tensile index of CNF-films by ~ 13%. By molecular modeling of fibril swelling, we found that water sorbed to cellulose exhibits a decreased diffusion constant compared to bulk water. We quantified this change and showed that diffusion of sorbed water displays less dependency on swelling temperature compared to bulk water diffusion. To our knowledge, this has not previously been demonstrated by molecular modeling. The difference between bulk water diffusion (DWW) and diffusion of water sorbed to cellulose (DCC) increased from DWW − DCC ~ 3 × 10–5 cm/s2 at 25 °C to DWW − DCC ~ 8.3 × 10–5 cm/s2 at 100 °C. Moreover, water molecules spent less successive time sorbed to a fibril at higher temperatures

A Brief Review of Nanocellulose Based Hybrid Membranes for CO₂ Separation

Due to the high specific surface area, high mechanical strength and broad possibility of surface modification, nanocellulose has obtained much attention as a new class of bio-based nanomaterials with promising potential in a wide variety of applications. Recently, a considerable amount of research has been aimed to the fabrication of nanocellulose based hybrid membranes for water treatment. However, nanocellulose based hybrid gas separation membrane is still a new research area. Herein, we force on recent advancements in the fabrication methods and separation performances of nanocellulose-based hybrid membranes for CO2 separation, the transport mechanisms involved, along with the challenges in the utilization of nanocellulose in membranes. Finally, some perspectives on future R&amp;D of nanocellulose-based membranes for CO2 separation are proposed

Mechanical properties of cellulose nanofibril films: effects of crystallinity and its modification by treatment with liquid anhydrous ammonia

The influence of cellulose crystallinity on mechanical properties of cellulose nano-fibrils (CNF) was investigated. Degree of crystallinity (DoC) was modified using liquid anhydrous ammonia. Such treatment changes crystal allomorph from cellulose I to cellulose III, a change which was reversed by subsequent boiling in water. DoC was measured using solid state nuclear magnetic resonance (NMR). Crystalline index (CI) was also measured using wide angle X-ray scattering (WAXS). Cotton linters were used as the raw material. The cotton linter was ammonia treated prior to fibrillation. Reduced DoC is seen to associate with an increased yield point and decreased Young modulus. Young modulus is here defined as the maximal slope of the stress–strain curves. The association between DoC and Young modulus or DoC and yield point are both statistically significant. We cannot conclude there has been an effect on strainability. While mechanical properties were affected, we found no indication that ammonia treatment affected degree of fibrillation. CNF was also studied in air and liquid using atomic force microscopy (AFM). Swelling of the nanofibers was observed, with a mean diameter increase of 48.9%