Range Of pHs Research Articles

An Orthogonal Workflow of Electrochemical, Computational, and Thermodynamic Methods Reveals Limitations of Using a Literature-Reported Insulin Binding Peptide in Biosensors.

Developing a continuous insulin-monitoring biosensor is of great importance for both the cellular biomanufacturing industry and for treating diabetes mellitus. Such a sensor needs to be able to effectively monitor insulin across a range of temperatures and pHs and with varying concentrations of competing analytes. One of the two main components of any biosensor is the recognition element, which is responsible for interacting with the molecule of interest. Prior literature describes an insulin-binding peptide (IBP) that was reported to bind to insulin with a 3 nM affinity. Here, we used orthogonal and complementary electrochemical, computational, and thermodynamic characterization methods to evaluate IBP's appropriateness for use in a biosensor. Unfortunately, all three methods failed to produce evidence of IBP-insulin binding either on surfaces or in solution. This indicates that the binding exhibited in previous reports is likely restricted to a limited set of conditions and that IBP is not a suitable recognition element for a continuous insulin biosensor.

Comparison of Colorimetric Methods for Measuring the Solubility of Legume Proteins.

Increasing the use of plant proteins in foods requires improving their physical and chemical properties, such as emulsification, gelation capacity, and thermal stability. These properties determine the acceptability and functionality of food products. Higher protein solubility significantly impacts these properties by affecting denaturation and the stability of emulsifiers or gels. Therefore, developing plant-based protein ingredients requires accurately and conveniently measuring their solubility. Colorimetric solubility methods overcome many issues of more robust combustion and titration methods, but complicated chemical mechanisms limit their applicability for certain proteins. This study aims to compare the effectiveness of four common colorimetric solubility measurement methods for pulse and non-pulse legume proteins and hydrolysates. Pea, chickpea, lentil, and soy protein isolates were made from defatted flour and their solubility at a range of pHs was measured using the Bradford, Lowry, bicinchoninic acid (BCA), and biuret methods. Solubility was also measured for chickpea and soy protein hydrolysates made using Alcalase and Flavourzyme. A comparison of the methods for solubility quantification revealed that the Bradford and Lowry methods most closely match the expected results for the unhydrolyzed protein, with the BCA and biuret methods underestimating solubility by 30%. The Lowry method was the preferred method for hydrolysate solubility measurement, with the Bradford method measuring 0% solubility at the isoelectric point due to an inability to interact with peptides that are soluble at this pH. This study identifies reliable methods for measuring plant protein solubility that establish uniform outcomes and enable a better comparison across studies, giving a consensus for key functional properties in food applications.

Exploring Mucoadhesive and Toxicological Characteristics Following Modification of Linear Polyethylenimine with Various Anhydrides.

Linear polyethylenimine (L-PEI) has numerous applications, such as in pharmaceutical formulations, gene delivery, and water treatment. However, due to the presence of secondary amine groups, L-PEI shows a relatively high toxicity and low biocompatibility. Here, various organic anhydrides were used to modify L-PEI to reduce its toxicity and enhance its functionality. We selected methacrylic anhydride, crotonic anhydride, maleic anhydride, and succinic anhydride to modify L-PEI. The structure of the resulting derivatives was characterized using 1H NMR and FTIR spectroscopies, and their behavior in aqueous solutions was studied using turbidimetric and electrophoretic mobility measurements over a broad range of pHs. A fluorescence flow through method determined the mucoadhesive properties of the polymers to the bovine palpebral conjunctiva. Methacrylated L-PEI and crotonylated L-PEI showed strong mucoadhesive properties at pH 7.4, likely due to covalent bonding with mucin thiol groups. In contrast, maleylated and succinylated L-PEI were poorly mucoadhesive as the pH was above their isoelectric point, resulting in electrostatic repulsion between the polymers and mucin. The toxicity of these polymers was evaluated using in vivo assays with planaria and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay in human alveolar epithelial cells. Moreover, the irritancy of polymers was assessed using a slug mucosa irritation assay. The results demonstrated that anhydride modification mitigated the adverse toxicity effects seen for parent L-PEI.

A novel cobalt-iron bimetallic hydrochar for the degradation of triclosan in the aqueous solution: performance, reusability, and synergistic degradation mechanism

Low activation performance is a critical issue limiting the practical application of low-cost biochar in the advanced oxidation. Given the high potential of transition metals in the persulfate activation process and abundant oxygen-containing groups of hydrochar, hydrochar derived from cobalt (Co)-modified iron (Fe)-enriched sludge was synthesized and its performance and activation mechanism for the degradation of triclosan were investigated. Co modification significantly altered the morphology of hydrochar, and the increased Co–Fe mass ratios transformed hydrochar from granular to rose-shaped lamellar and then to helical sheet structures. Specific surface area, defect degree, and oxygen-containing groups of hydrochar increased with increasing cobalt-iron mass ratios. The highest removal of triclosan was up to 98% in the hydrochar/peroxymonosulfate (PMS) system under a wide range of pHs (3–10) and still remained higher than 90% after four cycles. Both Radical (mainly hydroxyl radical) and nonradical pathways (singlet oxygen and electron transfer) were evidenced to play roles in the triclosan removal. Fe3+ promoted the regeneration of Co2+ and realized the efficient circulation of Co3+/Co2+. A ternary system consisting of electron donor (triclosan)-electron mediator (hydrochar)-electron acceptor (PMS) provided channels for electron transfer. No measurable Co and Fe were released during the reaction, and the toxicity of degradation intermediates was lower than that of triclosan. Beside triclosan, rhodamine B, bisphenol A, sulfamethoxazole, and phenol were also almost degraded completely in this oxidation system. This study provides a promising way for the enhancement of catalytic activity of carbonaceous material.

Freezing-induced acidification of sea ice brine

The acidity of sea ice and snow plays a key role in the chemistry of the cryosphere; an important example lies in the photochemical catalytic release of reactive bromine in polar regions, facilitated at pHs below 6.5. We apply in-situ acid-base indicators to probe the microscopic acidity of the brine within the ice matrix in artificial sea water at a range of concentrations (0.35–70 PPT) and initial pHs (6–9). The results are supported by analogous measurements of the most abundant salts in seawater: NaCl, Na2SO4, and CaCO3. In the research herein, the acidity is expressed in terms of the Hammett acidity function, H2−. The obtained results show a pronounced acidity increase in sea water after freezing at −15 °C and during the subsequent cooling down to −50 °C. Importantly, we did not observe any significant hysteresis; the values of acidity upon warming markedly resembled those at the corresponding temperatures at cooling. The acidity increase is attributed to the minerals' crystallization, which is accompanied by a loss of the buffering capacity. Our observations show that lower salinity sea water samples (≤ 3.5 PPT) reach pH values below 6.5 at the temperature of −15 °C, whereas higher salinity ices attain such values only at −30 °C. The ensuing implications for polar chemistry and the relevance to the field measurements are discussed.

MzmL, a novel marine derived N-acyl homoserine lactonase from Mesoflavibacter zeaxanthinifaciens that attenuates Pectobacterium carotovorum subsp. carotovorum virulence.

Quorum sensing (QS) is a conserved cell-cell communication mechanism widely distributed in bacteria, and is oftentimes tightly correlated with pathogen virulence. Quorum quenching enzymes, which interfere with QS through degrading the QS signaling molecules, could attenuate virulence instead of killing the pathogens, and thus are less likely to induce drug resistance. Many Gram-negative bacteria produce N-acyl homoserine lactones (AHLs) for interspecies communication. In this study, we isolated and identified a bacterial strain, Mesoflavibacter zeaxanthinifaciens XY-85, from an Onchidium sp. collected from the intertidal zone of Dapeng Reserve in Shenzhen, China, and found it had strong AHL degradative activity. Whole genome sequencing and blast analysis revealed that XY-85 harbors an AHL lactonase (designated MzmL), which is predicted to have an N-terminal signal peptide and share the "HXHXDH" motif with known AHL lactonases belonging to the Metallo-β-lactamase superfamily. Phylogenetic studies showed MzmL was closest to marine lactonase cluster members, MomL and Aii20J, instead of the AiiA type lactonases. Ultra performance liquid chromatography-mass spectrometry analysis confirmed that MzmL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MzmL could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, and retained full bioactivity under a wide range of temperatures (28-100°C) and pHs (4-11). Furthermore, MzmL significantly reduced Pectobacterium carotovorum subsp. carotovorum virulence factor production in vitro, such as biofilm formation and plant cell wall degrading enzyme production, and inhibited soft rot development on potato slices. These results demonstrated that MzmL may be a novel type of AHL lactonase with good environmental stability, and has great potential to be developed into a novel biological control agent for bacterial disease management.

Magnetic field-assisted adsorption of phosphate on biochar loading amorphous Zr–Ce (carbonate) oxide composite

For metal-based phosphate adsorbents, the dispersity and utilization of surface metal active sites are crucial factors in their adsorption performance and synthesis cost. In this study, a biochar material modified with amorphous Zr–Ce (carbonate) oxides (BZCCO-13) was synthesized for the phosphate uptake, and the adsorption process was enhanced by magnetic field. The beside-magnetic field was shown to have a better influence than under-magnetic field on adsorption, with maximum adsorption capacities (123.67 mg P/g) 1.14-fold greater than that without magnetic field. The beside-magnetic field could also accelerate the adsorption rate, and the time to reach 90% maximum adsorption capacity decreased by 83%. BZCCO-13 has a wide range of application pHs from 5.0 to 10.0, with great selectivity and reusability. The results of XPS and ELNES showed that the "magnetophoresis" of Ce3+ under the magnetic field was the main reason for the enhanced adsorption performance. In addition, increased surface roughness, pore size and oxygen vacancies, enhanced mass transfer by Lorentz force under a magnetic field, all beneficially influenced the adsorption process. The mechanism of phosphate adsorption by BZCCO-13 could be attributed to electrostatic attraction and CO32–dominated ligand exchange. This study not only provided an effective strategy for designing highly effective phosphate adsorbents, but also provides a new light on the application of rare earth metal-based adsorbent in magnetic field.

Improving the hydrostability of zeolitic imidazolate framework coatings using a facile silk fibroin protein modification method

Zeolitic imidazolate frameworks (ZIFs) are an important subclass of metal-organic frameworks (MOFs) with zeolite-type topology, which can be fabricated under ambient synthesis conditions. However, the applications of ZIFs are commonly limited due to the weak hydrostability of their metal–ligand coordination bonds, particularly under humid and aqueous conditions. In this work, as an example, the hydrolysis behaviours of ZIF-L with a special focus on ZIF-L coatings were tested at aqueous conditions with a wide range of pHs to systematically study and fundamentally understand their structural stability and degradation mechanism. Pristine ZIF-L powder and ZIF-L coatings were severely damaged after only 24 h in aqueous media. Interestingly, the ZIF-L coatings showed two distinct hydrolyzation pathways regardless of pH conditions, exhibiting either a ring-shaped etching or unfolding behaviours. While the ZIF-L powders were hydrolyzed almost identically across all pH conditions. With this new understanding, a facile silk fibroin (SF) protein modification method was developed to enhance the hydrostability of ZIF-L coatings in aqueous media. The effect of protein concentration on surface coating was systemically studied. ZIF-L coating retained its surface morphology after soaking in water and demonstrated switchable super wetting properties and superior separation performance for oil/water mixture. As a result, the quick SF protein modification significantly enhanced the stability of ZIF-L coatings under various pHs, while retaining their switchable wetting property and excellent separation performance.

Mitigation of Mycotoxins in Food-Is It Possible?

Among microorganisms found in food, fungi stand out because they are adaptable and competitive in a large range of water activities, temperatures, pHs, humidities and substrate types. Besides sporulating, some species are toxigenic and produce toxic metabolites, mycotoxins, under adverse biotic and abiotic variables. Microorganisms are inactivated along the food chain, but mycotoxins have stable structures and remain in ready-to-eat food. The most prevalent mycotoxins in food, which are aflatoxins, fumonisins, ochratoxin A, patulin, tenuazonic acid, trichothecenes and zearalenone, have maximum tolerable limits (MTLs) defined as ppb and ppt by official organizations. The chronic and acute toxicities of mycotoxins and their stability are different in a chemical family. This critical review aims to discuss promising scientific research that successfully mitigated levels of mycotoxins and focus the results of our research group on this issue. It highlights the application of natural antifungal compounds, combinations of management, processing parameters and emergent technologies, and their role in reducing the levels and bioaccessibility. Despite good crop management and processing practices, total decontamination is almost impossible. Experimental evidence has shown that exposure to mycotoxins may be mitigated. However, multidisciplinary efforts need to be made to improve the applicability of successful techniques in the food supply chain to avoid mycotoxins' impact on global food insecurity.

Probing the binding sites of bioactives with β-Lactoglobulin at different gastrointestinal pHs

Encapsulation is an efficient strategy to preserve the nutritional value of bioactive compounds in functional foods. The whey protein, β-lactoglobulin (βLg), is a potential carrier for bioactives due to its stability over a range of pHs and in the presence of the gastric enzyme, pepsin. In this study, curcumin and folic acid are used as representative hydrophobic and hydrophilic bioactives compounds respectively to investigate the suitability of βLg to encapsulate at different pH conditions of the gastrointestinal tract (2.0, 4.0, and 7.4). Unexpectedly, we found through intrinsic fluorescence spectroscopy that βLg binds both curcumin and folic acid with similar binding affinity at all three pHs. Fourier-transform infrared experiments showed, that with an exception at pH 2.0, H-bonding play a role in the interaction of both bioactives with βLg. Circular dichroism spectroscopy and small angle X-ray scattering showed that the interactions does not alter the structure of the protein. Further, 2D-protein nuclear magnetic resonance spectroscopy demonstrated, that curcumin binds to an external groove at pH 2.65. Whereas at pH 4.0, curcumin was found to bind at the groove and contrary to expectations, also at the calyx (the EF loop was previously thought to close the entrance to the calyx at this pH). At pH 6.5, both curcumin and folic acid bind to βLg at the calyx and the groove. Overall, our study shows that βLg binds curcumin and folic acid at gastrointestinal pHs, suggesting its potential ability to encapsulate both hydrophobic and hydrophilic bioactive molecules in dairy functional foods.

Biochemical characterization and gene structure analysisof the 24-kDa glutathione transferase sigma from Taenia solium.

Taenia solium can cause human taeniasis and/or cysticercosis. The latter can in some instances cause human neurocysticercosis which is considered a priority in disease-control strategies and the prevention of mental health problems. Glutathione transferases are crucial for the establishment and long-term survival of T. solium; therefore, we structurally analyzed the 24-kDa glutathione transferase gene (Ts24gst) of T. solium and biochemically characterized its product. The gene promoter showed potential binding sites for transcription factors and xenobiotic regulatory elements. The gene consists of a transcription start site, four exons split by three introns, and a polyadenylation site. The gene architecture is conserved in cestodes. Recombinant Ts24GST (rTs24GST) was active and dimeric. Anti-rTs24GST serum showed slight cross-reactivity with human sigma-class GST. A 3D model of Ts24GST enabled identification of putative residues involved in interactions of the G-site with GSH and of the H-site with CDNB and prostaglandin D2. Furthermore, rTs24GST showed optimal activity at 45 °C and pH 9, as well as high structural stability in a wide range of temperatures and pHs. These results contribute to the better understanding of this parasite and the efforts directed to fight taeniasis/cysticercosis.

Controlled release herbicide formulation for effective weed control efficacy

Controlled release formulation (CRF) of herbicide is an effective weed management technique with less eco-toxicity than other available commercial formulations. To maximise the effectiveness of CRFs however, it is crucial to understand the herbicide-releasing behaviour at play, which predominately depends on the interaction mechanisms between active ingredients and carrier materials during adsorption. In this study, we investigated and modelled the adsorption characteristics of model herbicide 2,4-D onto two organo-montmorillonites (octadecylamine- and aminopropyltriethoxysilane-modified) to synthesise polymer-based CRFs. Herbicide-releasing behaviour of the synthesised CRF microbeads was then analysed under various experimental conditions, and weed control efficacy determined under glasshouse conditions. Results revealed that adsorption of 2,4-D onto both organo-montmorillonites follows the pseudo-second-order kinetics model and is predominately controlled by the chemisorption process. However, multi-step mechanisms were detected in the adsorption on both organoclays, hence intra-particle diffusion is not the sole rate-limiting step for the adsorption process. Both organoclays followed the Elovich model, suggesting they have energetically heterogeneous surfaces. Herbicide-releasing behaviours of synthesised beads were investigated at various pH temperatures and ionic strengths under laboratory and glasshouse conditions. Furthermore, weed control efficacy of synthesised beads were investigated using pot studies under glasshouse condition. Desorption studies revealed that both synthesised microbeads have slow releasing behaviour at a wide range of pHs (5–9), temperatures (25–45 °C), and ionic strengths. The results also revealed that synthesised microbeads have excellent weed control efficacy on different broad-leaf weed species under glasshouse conditions.

Effect of seawater temperature and pH on the sperm motility of the European eel.

The current climate change situation could bring critical effects for marine species, especially those already considered endangered. Although some species can adapt fast to the environmental changes, it is necessary to get into the worst scenario and develop tools to anticipatedly assess the physiological effects of such environmental change. With this purpose, our study aims to determine the effect of a range of seawater temperatures and pHs on sperm motility in the European eel (Anguilla anguilla). Low seawater pH (6.5-7.4) decreased the eel sperm motility in comparison to the control (pH = 8.2). We also studied the combined effect of the pH of the artificial seminal plasma (the plasma where the sperm cells are suspended) with the pH of Artificial Sea Water (ASW, pH 7.8 or and 8.2). We did not find statistical differences in sperm motility and kinetic parameters caused by the artificial seminal plasma pH. However, seawater pH induced significantly higher values of total sperm motility, and the percentage of fast spermatozoa with a pH of 8.2 in comparison with a pH of 7.8. In contrast, the seawater temperature did not affect sperm motility parameters or sperm longevity. To study the effect of the interaction between seawater temperature and pH on sperm motility, two temperatures: 4 and 24°C, and two pHs 7.8 and 8.2, were tested. There were significant differences between temperature and pH in several kinetic parameters and, in general, the lowest values were observed in the samples activated at low temperature and low pH (4°C, pH 7.8). This work suggest that eel sperm motility and kinetics will not be affected by the expected changes in pH or temperature due to the climate change.

IDENTIFYING THE ROLE OF ACINETOBACTER CALCOACETICUS IN DRIVING INTESTINAL INFLAMMATION

Abstract BACKGROUND Inflammatory bowel disease (IBD) is a life-long condition characterized by chronic inflammation of the gastrointestinal tract. Among the potential causes of IBD, the gut microbiota has been implicated in perpetuating inflammation. Analysis of the IBD Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) database revealed that Acinetobacter calcoaceticus is one of the top 10 highest elevated bacteria in Crohn’s’ disease patients. Acinetobacter are well-characterized for their antibiotic resistance, but little is known about their interaction with the gut. We hypothesize that A. calcoaceticus colonizes the gut and promotes intestinal inflammation. MATERIALS & RESULTS We grew commercially available and clinical isolates of A. calcoaceticus in vitro with stressors found in the gut. All strains grew in a range of pHs (4-7), osmolarity (0.1-1 M NaCl), ethanol (1- 5%) and hydrogen peroxide (0.05-0.1%); indicating that A. calcoaceticus is well-adapted to withstand the harsher conditions of the gastrointestinal tract. To further investigate the ability of A. calcoaceticus to colonize the gut, we utilized Caenorhabditis elegans as a model. All A. calcoaceticus strains colonized the nematode's gut. We likewise found that all A. calcoaceticus strains colonized human stool-based bioreactors; suggesting that A. calcoaceticus can colonize the gastrointestinal tract. To identify whether A. calocaceticus stimulated inflammation, we incubated intestinal organoids and macrophages with live A. calcoaceticus or LPS derived from our A. calcaoceticus strains and found that both conditions stimulated pro-inflammatory cytokines. In order to confirm our in vitro findings, we oral gavaged adult BalbC mice with A. calcoaceticus and examined fecal levels using selective agar and qPCR. Acinetobacter was observed at low levels in the vehicle control mice and at high levels in our mice gavaged with A. calcoaceticus (average 9.9x 107 CFU); suggesting that A. calcoaceticus colonized the murine gut. To examine the ability of A. calcoaceticus to enhance inflammation, we oral gavaged mice with either a vehicle control (PBS) or A. calcoaceticus and the following day we administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. Mice treated with A. calcoaceticus and TNBS lost more weight and had worse histological scores than mice treated with vehicle control and TNBS; indicating A. calcoaceticus worsens intestinal inflammation. Finally, to expand the niche of Acinetobacter, we pre-treated mice with antibiotics and then subsequently administered bacteria and TNBS. In this model, A. calcoaceticus exacerbated inflammation above that of non-antibiotic treated mice. CONCLUSIONS These data show that A. calcoaceticus can promote intestinal inflammation and may be a contributing factor to the inflammation experienced by IBD patients.

Ultrarapid and Sustainable Synthesis of Trimetallic-Based MOF (CrNiFe-MOF) from Stainless Steel and Disodium Terephthalate-Derived PET Wastes.

Designing easy and sustainable strategies for the synthesis of metal-organic frameworks (MOFs) from organic and inorganic wastes with the efficient removal of phosphate from water remains a challenge. The majority of the reported works have utilized costly precursors and nonsoluble ligands for the synthesis of MOFs. Herein, we have developed a low-cost, simple, and sustainable alternative approach using the coprecipitation method in water at room temperature for the synthesis of a new adsorbent-based trimetallic MOF. Poly(ethylene terephthalate) and stainless steel wastes were used as sources of water-soluble disodium terephthalate ligand and three metallic species (chromium, nickel, and iron salts) for the fabrication of trimetallic MOF (CrNiFe-MOF), respectively. The newly developed MOF demonstrates a superior space-time yield of 5760 g m-3 day-1, reaching a level allowing the industrialization production of this sustainable MOF. The scanning electron microscopy and adsorption studies revealed that the developed trimetallic MOF consists of aggregated nanoparticles and the presence of defective as well as mesoporous structures. This MOF showed an enhanced adsorption capacity of phosphate from real eutrophic water samples and higher stability in a range of pHs. The density functional theory calculations evidenced that the phosphate ions preferentially adsorb over H2O toward the metal oxo-trimers, with the adsorption energies increasing from H3PO4 to PO43- species in line with an improvement of the adsorption performance of CrNiFe-MOF when the pH increases, i.e., when HPO42- and PO43- become more predominant. These calculations also supported that the incorporation of Cr metal sites in the oxo-trimer is expected to boost the phosphate affinity of the MOF. Finally, our work provides an easy and eco-friendly approach for MOF designing to enhance phosphate removal from water.

On the mucoadhesive properties of synthetic and natural polyampholytes

HypothesisThe mucoadhesive characteristics of amphoteric polymers (also known as polyampholytes) can vary and are influenced by factors such as the solution's pH and its relative position against their isoelectric point (pHIEP). Whilst the literature contains numerous reports on mucoadhesive properties of either cationic or anionic polymers, very little is known about these characteristics for polyampholytes ExperimentsHere, two amphoteric polymers were synthesized by reaction of linear polyethylene imine (l-PEI) with succinic or phthalic anhydride and their mucoadhesive properties were compared to bovine serum albumin (BSA), selected as a natural polyampholyte. Interactions between these polymers and porcine gastric mucin were studied using turbidimetric titration and isothermal titration calorimetry across a wide range of pHs. Model tablets were designed, coated with these polymers and tested to evaluate their adhesion to porcine gastric mucosa at different pHs. Moreover, a retention study using fluorescein isothiocyanate (FITC)-labelled polyampholytes deposited onto mucosal surfaces was also conducted FindingsAll these studies indicated the importance of solution pH and its relative position against pHIEP in the mucoadhesive properties of polyampholytes. Both synthetic and natural polyampholytes exhibited strong interactions with mucin and good mucoadhesive properties at pH < pHIEP.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-β-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two β-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-β-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-β-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Effective Isolation and Characterization of Mycobacteriophages with the Ability to Lyse Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD), a chronic infectious enteritis of ruminants, causes major economic losses in the dairy industry globally. This enteritis is caused by Mycobacterium avium subsp. Paratuberculosis (MAP). Currently there is no cure for JD and test-based culling has proved ineffective at preventing the spread. To isolate new mycobacteriophages (mbps) that can potentially be used to control JD transmission and infection on dairy farms, we optimized an isolation protocol by fecal spiking and the testing of different isolation solution compositions. Using this protocol, we successfully enhanced the yield of mbps from spiked fecal samples, elevating it from less than 1% to 59%. With this method, we isolated 14 mbps from 475 environmental samples collected from MAP-positive dairy farms, after in-sample enrichment with MAP and the fast-growing M. smegmatis. The sample sources included soil, manure pits, lactation barns, feces, milk, and drain water. After fingerprinting these mbps by restriction enzyme profiling, we concluded that 12 were distinct and novel. Further characterization of their host range revealed that eight were capable of lysing multiple MAP strains. We also studied the cross-resistance, lysogeny, the effect of pH and their antimycobacterial properties in milk replacer. Each novel mbp showed limited cross-resistance and prophage immunity and showed no reduction in the titer in a range of pHs after 4 h. The novel phages were also able to reduce the mycobacterial counts to zero after 8 h in milk replacer. In conclusion, these novel mbps could be considered to be used in the control strategies of JD on farms.

Arsenic sorption and oxidation by natural manganese-oxide-enriched soils: Reaction kinetics respond to varying environmental conditions

Manganese-oxides are some of the strongest oxidants and sorbents in the environment, which impact many geochemical processes. However, nearly all our understanding of manganese-oxides’ reaction kinetics is based on laboratory-synthesized minerals. This study quantifies the oxidative kinetics and adsorptive capacity of five soils rich in pedogenic manganese- and iron-oxides through arsenite oxidation batch reactions over a range of pHs and temperatures to mimic diverse environmental conditions. The two A horizons were less reactive and enriched in manganese(IV), compared to the B horizons, particularly the subsoil containing the manganese-rich wad material. The reaction kinetics fit a pseudo-first-order reaction with distinct fast and slow phases. The baseline reactions were pH 7.2 at 23 °C. Adjusting pH to 4.5 or 9.0 increased the reaction rates. Decreasing the temperature to 4.0 °C reduced the reaction kinetics, while raising the temperature to 40 °C increased the arsenite oxidation rate. pH and temperature changes alter the reaction kinetics due to shifts related to the point of zero charge, the total system energy, and surface passivation from adsorbing arsenic and manganese species. Synchrotron X-ray fluorescence mapping indicates arsenic only penetrates the surficial layers of most manganese-oxide-containing nodules found in the soil. After the arsenite oxidation reaction in the pedogenically weathered subsoil, X-ray absorption spectroscopy demonstrates significant differences in the average manganese oxidation number between the nodules’ outer layers compared to the soil matrix and nodule centers. The kinetic and sorption parameters give critical insight into determining the mobility and species of arsenic and other redox-sensitive contaminants in manganese-containing environmental systems over appropriate timescales.

Structural, rheological properties and antioxidant activities analysis of the exopolysaccharide produced by Rhizobium leguminosarum bv. viciae VF39

Microbial exopolysaccharide is an eco-friendly and non-toxic biopolymeric materials widely used in various industrial fields such as pharmaceutical, food and cosmetics based on its structural, rheological and physiochemical properties. A microbial exopolysaccharide (VF39-EPS) was directly isolated from Rhizobium leguminosarum bv. viciae VF39. Structural analysis using FTIR and 2D NMR spectroscopy confirmed the complete chemical structures of VF39-EPS as 3-hydroxybutanoylglycan with octasaccharide repeating units containing two pyruvyl, two acetyl, and one 3-hydroxybutanoyl group. VF39-EPS exhibited thermal stability up to 275 °C and showed characteristic rheological behaviors of structural fluid with weak gel-like properties above 4 % the aqueous solution, suggesting VF39-EPS as a potential effective thickener or hydrogel scaffolder. Flow behavior tests validated broad stability at a wide range of both pHs from 2 to 12 and temperatures from 25 to 75 °C, and even in the presence of various salts. Furthermore, VF39-EPS showed excellent antioxidant effects of 78.5 and 62.4 % (n = 3, p < 0.001) in DPPH scavenging activity and hydroxyl radical scavenging activity, respectively. Therefore, those structural, rheological and antioxidant properties suggest that VF39-EPS could be one of the excellent biomaterial candidates for cosmetic, food and pharmaceutical industries based on its characteristic rheological behaviors in various condition and excellent antioxidant activity.