Expression of Biofilm-Degrading Enzymes in Plants and Automated High-Throughput Activity Screening Using Experimental Bacillus subtilis Biofilms

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

Asymmetric depth-filtration - a versatile and scalable approach for isolation and purification of extracellular vesicles

A novel asymmetric depth filtration (DF) approach for isolation of extracellular vesicles (EVs) from biological fluids is presented, and its performance is compared with established methods. The developed workflow is simple, inexpensive, and relatively fast. Compared with ultracentrifugation and size-exclusion chromatography, the developed method isolates EVs with higher purity and yield. Only standard laboratory equipment is needed for its implementation, which makes it suitable for low-resource locations. The described implementation of the method is suitable for EV isolation from small biological samples in diagnostic and treatment guidance applications. Following the scale-up routes adopted in the biomanufacturing of therapeutics, which routinely rely on DF as one of the product purification steps, the developed method may be scaled up to harvesting therapeutic EVs from large volumes of growth medium.

Simplified method for the rapid quantification of copper fractions in wine

Simple and inexpensive methodologies for quantifying the total Cu concentration as well as various Cu fractions in wine have been recently developed, including the application of colorimetric analysis, and/or diatomaceous earth depth filtration. Compared to other conventional Cu analysis methods, the proposed approaches are more suitable for winery laboratories for reliable and affordable quantification of total Cu and its fractions during wine production.

Hybrid Sand Control Screen Using the Combined Surface and Depth Filtration

Development of weakly and unconsolidated sand reservoirs require effective sand control media to prevent sand production. The existing sand control devices in the market are either relying on surface filtration to prevent sand production through size exclusion or bridging or depth filtration which relies on the pore size distribution of a porous filter or pack to prevent the sand from producing along the production fluids. In this study, we introduce a new hybrid sand screen that works based on a combined surface and depth filtration. Radial Sand Control Evaluation (RSCE) testing facility was used to compare the solid production and flow performance of the new hybrid screen with various mesh media in multi-phase gas and liquid flow under various fluid injection scenarios. Solid production and flow performance were compared with investigated cases. The new hybrid screen provides an optimized Open to Flow Area (OFA) in comparison to available surface filtration or depth filtration media, which provides required OFA, while prevents sanding. The robust design, low cost and manufacturing ease make it a suitable screen media for most sand control applications. The sand retention test results under various fluid injection scenarios including multi-phase oil, brine, and gas show that it outperforms the Dutch Twill (DT) weave and Reverse Dutch Twill (RDT) weave of equivalent aperture size, with better flow performance at constant flow rate tests compare to best-performing mesh media, while keeping the produced sand far below the acceptable thresholds. Hybrid design handles both high velocity and high Gas-Oil Ratio (GOR) better than equivalent depth filtration media of equivalent size. This paper presents a detailed characterization, flow performance testing of a new hybrid sand control media that combines the surface filtration and depth filtration properties to achieve better solid retention and flow performance. The hybrid screen media is suitable for high-rate producers with high GOR. Keywords: Hybrid Screen, Surface Filtration, Depth Filtration, Radial Sand Control Evaluation (RSCE) Testing

Depth-graded motivic multiple zeta values

We study the depth filtration on multiple zeta values, on the motivic Galois group of mixed Tate motives over $\mathbb {Z}$ and on the Grothendieck–Teichmüller group, and its relation to modular forms. Using period polynomials for cusp forms for $\mathrm {SL} _2(\mathbb {Z})$, we construct an explicit Lie algebra of solutions to the linearized double shuffle equations, which gives a conjectural description of all identities between multiple zeta values modulo $\zeta (2)$ and modulo lower depth. We formulate a single conjecture about the homology of this Lie algebra which implies conjectures due to Broadhurst and Kreimer, Racinet, Zagier, and Drinfeld on the structure of multiple zeta values and on the Grothendieck–Teichmüller Lie algebra.

Silicon carbide (SiC) membranes in œnology: a laboratory-scale study

Unfiltered wine is a turbid medium that is not generally accepted by the consumer. Therefore, one or several filtration steps are required before bottling. Silicon carbide (SiC) membranes desirable parameters (porosity, tortuosity fluxes) allow filtering several different types of loaded matrices like wine or residue sediment. An in-depth filtration study was carried out on white and red wines to evaluate membrane efficiency and to optimise their cleaning procedure. Retention rates were studied as a function of wine type, filtration mode, and volumetric concentration factor. Compared to ceramic membranes, SiC membrane permeate fluxes are higher, up to a factor of 10 for red wine. For white wines, equivalent permeate fluxes could be obtained with dead-end filtration. Moreover, SiC membranes appear to be effective in obtaining a clear and brilliant wine and do not modify the concentration of the compounds of interest in wine. Finally, an optimised cleaning protocol has been identified and shown to restore a sufficient permeability to the SiC membranes.