Ramona Valentina Mateiu

Novel Aggregative Adherence Fimbria Variant of Enteroaggregative Escherichia coli

ABSTRACT Enteroaggregative Escherichia coli (EAEC) organisms belong to a diarrheagenic pathotype known to cause diarrhea and can be characterized by distinct aggregative adherence (AA) in a stacked-brick pattern to cultured epithelial cells. In this study, we investigated 118 EAEC strains isolated from the stools of Danish adults with travelers diarrhea. We evaluated the presence of the aggregative adherence fimbriae (AAFs) by a multiplex PCR, targeting the four known major subunit variants as well as their usher-encoding genes. Almost one-half (49/118) of the clinical isolates did not possess any known AAF major fimbrial subunit, despite the presence of other AggR-related loci. Further investigation revealed the presence of an AAF-related gene encoding a yet-uncharacterized adhesin, termed agg5A. The sequence of the agg5DCBA gene cluster shared fimbrial accessory genes (usher, chaperone, and minor pilin subunit genes) with AAF/III, as well as the signal peptide present in the beginning of the agg3A gene. The complete agg5DCBA gene cluster from a clinical isolate, EAEC strain C338-14, with the typical stacked-brick binding pattern was cloned, and deletion of the cluster was performed. Transformation to a nonadherent E. coli HB101 and complementation of the nonadherent C338-14 mutant with the complete gene cluster restored the AA adhesion. Overall, we found the agg5A gene in 12% of the 118 strains isolated from Denmark, suggesting that this novel adhesin represents an important variant.

Cascade catalysis in membranes with enzyme immobilization for multi-enzymatic conversion of CO2 to methanol

Facile co-immobilization of enzymes is highly desirable for bioconversion methods involving multi-enzymatic cascade reactions. Here we show for the first time that three enzymes can be immobilized in flat-sheet polymeric membranes simultaneously or separately by simple pressure-driven filtration (i.e. by directing membrane fouling formation), without any addition of organic solvent. Such co-immobilization and sequential immobilization systems were examined for the production of methanol from CO2 with formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Enzyme activity was fully retained by this non-covalent immobilization strategy. The two immobilization systems had similar catalytic efficiencies because the second reaction (formic acid→formaldehyde) catalyzed by FaldDH was found to be the cascade bottleneck (a threshold substrate concentration was required). Moreover, the trade-off between the mitigation of product inhibition and low substrate concentration for the adjacent enzymes probably made the co-immobilization meaningless. Thus, sequential immobilization could be used for multi-enzymatic cascade reactions, as it allowed the operational conditions for each single step to be optimized, not only during the enzyme immobilization but also during the reaction process, and the pressure-driven mass transfer (flow-through mode) could overcome the diffusion resistance between enzymes. This study not only offers a green and facile immobilization method for multi-enzymatic cascade systems, but also reveals the reaction bottleneck and provides possible solutions for the bioconversion of CO2 to methanol.

Functionalization of a Membrane Sublayer Using Reverse Filtration of Enzymes and Dopamine Coating

High permeability, high enzyme loading, and strong antifouling ability are the desired features for a biocatalytic membrane to be used in an enzymatic membrane reactor (EMR). To achieve these goals, the membrane sublayer was enriched with laccase by reverse filtration in this case, and the resulting enzyme-loaded sublayer was covered with a dopamine coating. After membrane reversal, the virgin membrane skin layer was facing the feed and the enzymes were entrapped by a polydopamine network in the membrane sublayer. Thus, the membrane sublayer was functionalized as a catalytically active layer. The effects of the original membrane properties (i.e., materials, pore size, and structure), enzyme type (i.e., laccase and alcohol dehydrogenase), and coating conditions (i.e., time and pH) on the resulting biocatalytic membrane permeability, enzyme loading, and activity were investigated. Using a RC10 kDa membrane with sponge-like sublayer to immobilize laccase with dopamine coating, the trade-off between permeability and enzyme loading was broken, and enzyme loading reached 44.5% without any permeability loss. After 85 days of storage and reuse 14 times, more than 80% of the immobilized laccase activity was retained for the membrane with a dopamine coating, while the relative activity was less than 40% without the coating. The resistance to high temperature and acidic/alkaline pH was also improved by the dopamine coating for the immobilized laccase. Moreover, this biocatalytic membrane could resist mild hydrodynamic cleaning (e.g., back-flushing), but the catalytic ability was reduced by chemical cleaning at extreme pH (e.g., 1.5 and 11.5). Since the immobilized enzyme is not directly facing the bulk of EMRs and the substrate can be specifically selected by the separation skin layer, this biocatalytic membrane is promising for cascade catalytic reactions.

Internal Porosity of Mineral Coating Supports Microbial Activity in Rapid Sand Filters for Groundwater Treatment

ABSTRACT A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH4 + removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 μm) of the mineral coating, which had a thickness of up to 600 ± 51 μm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.

Peptoids successfully inhibit the growth of gram negative E. coli causing substantial membrane damage

Peptoids are an alternative approach to antimicrobial peptides that offer higher stability towards enzymatic degradation. It is essential when developing new types of peptoids, that mimic the function of antimicrobial peptides, to understand their mechanism of action. Few studies on the specific mechanism of action of antimicrobial peptoids have been described in the literature, despite the plethora of studies on the mode of action of antimicrobial peptides. Here, we investigate the mechanism of action of two short cationic peptoids, rich in lysine and tryptophan side chain functionalities. We demonstrate that both peptoids are able to cause loss of viability in E. coli susceptible cells at their MIC (16–32 μg/ml) concentrations. Dye leakage assays demonstrate slow and low membrane permeabilization for peptoid 1, that is still higher for lipid compositions mimicking bacterial membranes than lipid compositions containing Cholesterol. At concentrations of 4 × MIC (64–128 μg/ml), pore formation, leakage of cytoplasmic content and filamentation were the most commonly observed morphological changes seen by SEM in E. coli treated with both peptoids. Flow cytometry data supports the increase of cell size as observed in the quantification analysis from the SEM images and suggests overall decrease of DNA per cell mass over time.

Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

Photobacterium galatheae sp. nov., a bioactive bacterium isolated from a mussel in the Solomon Sea

A novel, Gram-negative marine bacterium, S2753T, was isolated from a mussel of the Solomon Sea, Solomon Islands. Analysis of the 16S rRNA gene sequence and whole genome sequence data placed strain S2753T in the genus Photobacterium with the closest relative being Photobacterium halotolerans DSM 18316T (97.7 % 16S rRNA gene similarity). Strain S2753T was able to grow from 15 to 40 °C and in NaCl concentrations of 0.5 to 9 % (w/v). The predominant fatty acids were 16 : 1ω7c/16 : 1ω6c (27.9 %), 16 : 0 (22.1 %) and 18 : 1ω7c/8 : 1ω6c (21.4 %). The genomic DNA G+C mol content was 49.5 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic differences, strain S2753T is considered to represent a novel species of the genus Photobacterium. Furthermore, whole genome sequence analysis comparing S2753T and type-strains of closely related species of the genus Photobacterium also demonstrated that the strain is genomically distinct enough to be considered a novel species. The name Photobacterium galatheae is proposed and the type-strain is S2753T( = LMG 28894T = DSM 100496T).

In Situ Formation of a Biocatalytic Alginate Membrane by Enhanced Concentration Polarization

A thin alginate layer induced on the surface of a commercial polysulfone membrane was used as a matrix for noncovalent immobilization of enzymes. Despite the expected decrease of flux across the membrane resulting from the coating, the initial hypothesis was that such a system should allow high immobilized enzyme loadings, which would benefit from the decreased flux in terms of increased enzyme/substrate contact time. The study was performed in a sequential fashion: first, the most suitable types of alginate able to induce a very thin, sustainable gel layer by pressure-driven membrane filtration were selected and evaluated. Then, an efficient method to make the gel layer adhere to the surface of the membrane was developed. Finally, and after confirming that the enzyme loading could remarkably be enhanced by using this method, several strategies to increase the permeate flux were evaluated. Alcohol dehydrogenase (EC 1.1.1.1), able to catalyze the conversion of formaldehyde into methanol, was selected as the model enzyme. An enzyme loading of 71.4% (44.8 μg/cm(2)) was attained under the optimal immobilization conditions, which resulted in a 40% conversion to methanol as compared to the control setup (without alginate) where only 10.8% (6.9 μg/cm(2)) enzyme was loaded, with less than 5% conversion. Such conversion increased to 60% when polyethylene glycol (PEG) was added during the construction of the gel layer, as a strategy to increase flux. No enzyme leakage was observed for both cases (with/without PEG addition). Modeling results showed that the dominant fouling mechanism during gel layer induction (involving enzyme entrapment) was cake layer formation in the initial and intermediate phases, while pore blocking was the dominant mechanism in the final phase. Such mechanisms had a direct consequence on the type of immobilization promoted in each phase. The results suggested that the strategy proposed could be efficiently used to enhance the enzyme loading on polymer membranes.

Nanoscale soldering of positioned carbon nanotubes using highly conductive electron beam induced gold deposition

We have developed an in-situ method for controlled positioning of carbon nanotubes followed by highly conductive contacting of the nanotubes, using electron beam assisted deposition of gold. The positioning and soldering process takes place inside an Environmental Scanning Electron Microscope (E-SEM) in the presence of a source of gold-organic precursor gas. Bridges deposited between suspended microelectrodes show resistivities down to 10/sup -4/ /spl Omega/cm and Transmission Electron Microscopy (TEM) of the deposits reveals a dense core of gold particles surrounded by a crust of small gold nanoparticles embedded in a carbon matrix. Nanoscale soldering of multi-walled carbon nanotubes (MWNT) onto microelectrodes was achieved by deposition of a conducting gold line across a contact point between nanotube and electrode. The solderings were found to be mechanically stronger than the carbon nanotubes. We have positioned MWNTs to bridge the gap between two electrodes, and formed soldering bonds between the tube and each of the electrodes. All nanotube bridges showed ohmic resistances in the range 10-30 k/spl Omega/. We observed no increase in resistance after exposing the MWNT bridge to air for days.

Use of Electrohydrodynamic Processing for Encapsulation of Sensitive Bioactive Compounds and Applications in Food

The use of vitamins, polyphenolic antioxidants, omega-3 polyunsaturated fatty acids (PUFAs), and probiotics for the fortification of foods is increasing. However, these bioactive compounds have low stability and need to be protected to avoid deterioration in the food system itself or in the gastrointestinal tract. For that purpose, efficient encapsulation of the compounds may be required. Spray drying is one of the most commonly used encapsulation techniques in the food industry, but it uses high temperature, which can lead to decomposition of the bioactive compounds. Recently, alternative technologies such as electrospraying and electrospinning have received increasing attention. This review presents the principles of electrohydrodynamic processes for the production of nano-microstructures (NMSs) containing bioactive compounds. It provides an overview of the current use of this technology for encapsulation of bioactive compounds and discusses the future potential of the technology. Finally, the review discusses advanced microscopy techniques to study the morphology of NMSs.