Stephanie Butler Velegol

Antimicrobial sand via adsorption of cationic Moringa oleifera protein.

Moringa oleifera (Moringa) seeds contain a natural cationic protein (MOCP) that can be used as an antimicrobial flocculant for water clarification. Currently, the main barrier to using Moringa seeds for producing potable water is that the seeds release other water-soluble proteins and organic matter, which increase the concentration of dissolved organic matter (DOM) in the water. The presence of this DOM supports the regrowth of pathogens in treated water, preventing its storage and later use. A new strategy has been established for retaining the MOCP protein and its ability to clarify and disinfect water while removing the excess organic matter. The MOCP is first adsorbed and immobilized onto sand granules, followed by a rinsing step wherein the excess organic matter is removed, thereby preventing later growth of bacteria in the purified water. Our hypotheses are that the protein remains adsorbed onto the sand after the functionalization treatment, and that the ability of the antimicrobial functionalized sand (f-sand) to clarify turbidity and kill bacteria, as MOCP does in bulk solution, is maintained. The data support these hypotheses, indicating that the f-sand removes silica microspheres and pathogens from water, renders adhered Escherichia coli bacteria nonviable, and reduces turbidity of a kaolin suspension. The antimicrobial properties of f-sand were assessed using fluorescent (live-dead) staining of bacteria on the surface of the f-sand. The DOM that can contribute to bacterial regrowth was shown to be significantly reduced in solution, by measuring biochemical oxygen demand (BOD). Overall, these results open the possibility that immobilization of the MOCP protein onto sand can provide a simple, locally sustainable process for producing storable drinking water.

The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

Moringa oleifera Seed Protein Adsorption to Silica: Effects of Water Hardness, Fractionation, and Fatty Acid Extraction

Motivated by the proposed use of cationic protein-modified sand for water filtration in developing nations, this study concerns the adsorption of Moringa oleifera seed proteins to silica surfaces. These proteins were prepared in model waters of varying hardness and underwent different levels of fractionation, including fatty acid extraction and cation exchange chromatography. Adsorption isotherms were measured by ellipsometry, and the zeta potentials of the resulting protein-decorated surfaces were measured by the rotating disk streaming potential method. The results indicate that the presence of fatty acids has little effect on the M. oleifera cationic protein adsorption isotherm. Adsorption from the unfractionated extract was indistinguishable from that of the cationic protein isolates at low concentrations but yielded significantly greater extents of adsorption at high concentrations. Adsorption isotherms for samples prepared in model hard and soft fresh waters were indistinguishable from each other over the measured bulk solution concentration range, but adsorption from hard or soft water was more extensive than adsorption from deionized water at moderate protein concentrations. Streaming potential measurements showed that adsorption reversed the net sign of the zeta potential of silica from negative to positive for all protein fractions and water hardness conditions at protein bulk concentrations as low as 0.03 μg/mL. This suggests that sands can be effectively modified with M. oleifera proteins using small amounts of seed extract under various local water hardness conditions. Finally, ellipsometry indicated that M. oleifera proteins adsorb irreversibly with respect to rinsing in these model fresh waters, suggesting that the modified sand would be stable on repeated use for water filtration. These studies may aid in the design of a simple, effective, and sustainable water purification device for developing nations.