Assessment of cryogenic pretreatment for simulating environmental weathering in the formation of surrogate micro- and nanoplastics from agricultural mulch film.

Microplastics (MPs) and nanoplastics (NPs) from mulch films and other plastic materials employed in vegetable and small fruit production pose a major threat to agricultural ecosystems. For conducting controlled studies on MPs' and NPs' (MNPs') ecotoxicity to soil organisms and plants and fate and transport in soil, surrogate MNPs are required that mimic MNPs that form in agricultural fields. We have developed a procedure to prepare MPs from plastic films or pellets using mechanical milling and sieving, and conversion of the resultant MPs into NPs through wet grinding, both steps of which mimic the degradation and fragmentation of plastics in nature. The major goal of this study was to determine if cryogenic exposure of two biodegradable mulch films effectively mimics the embrittlement caused by environmental weathering in terms of the dimensional, thermal, chemical, and biodegradability properties of the formed MNPs. We found differences in size, surface charge, thermal and chemical properties, and biodegradability in soil between MNPs prepared from cryogenically treated vs. environmentally weathered films, related to the photochemical reactions occurring in the environment that were not mimicked by cryogenic treatment, such as depolymerization and cross-link formation. We also investigated the size reduction process for NPs and found that the size distribution was bimodal, with populations centered at 50 nm and 150-300 nm, and as the size reduction process progressed, the former subpopulation's proportion increased. The biodegradability of MPs in soil was greater than for NPs, a counter-intuitive trend since greater surface area exposure for NPs would increase biodegradability. The result isassociated with differences in surface and chemical properties and to minor components that are readily leached out during the formation of NPs. In summary, the use of weathered plastics as feedstock would likely produce MNPs that are more realistic than cryogenically-treated unweathered films for use in experimental studies.

Melittin exerts opposing effects on short- and long-range dynamics in bicontinuous microemulsions.

Bicontinuous microemulsions (BµEs) are a promising biomembrane mimetic system for investigating the behavior of antimicrobial peptides (AMPs) and their delivery to open wounds to combat antibiotic-resistant microorganisms. The properties of the BµE host are in turn affected by the guest AMP and can deviate from those of the unperturbed BµEs, especially at higher AMP concentrations. Here we report the effect of an archetypal AMP, melittin, over a wide range of concentrations, on the nanoscopic dynamics of BµEs formed by water/sodium dodecyl sulfate (SDS)/1-pentanol/dodecane, investigated using quasi-elastic neutron scattering (QENS). Two distinct motions are observed, namely, (i) the lateral motion of the surfactant on the surface of the oil channels and (ii) the internal motion of the surfactants. It is found that melittin restricts both the lateral and the internal motion, thereby acting as a stiffening agent. The lateral motion is more strongly affected, at low concentration of melittin. The lateral diffusion coefficient decreased sharply, approaching a constant value at higher melittin concentration. These results are in sharp contrast with the recent dynamic light scattering and neutron spin echo results which showed that at the length and time scales longer than those probed in the current work, melittin enhanced the long-range collective and local undulation motions of BµEs. Considered together, our results indicate that incorporation of melittin modulates the dynamics differently depending on the spatial and temporal regimes, in which the dynamics are being probed. The addition of melittin at low concentrations increased the magnitude of the zeta potential, but further increase of the melittin concentration decreased it. This suggests that addition of melittin at low concentrations led to increase in the surfactant concentration, but did not affect the negative charge per surfactant molecule, while further addition of melittin led to ion pairing of melittin with the oppositely charged surfactant. This study therefore demonstrates how the addition of melittin hinders the lateral motion of surfactants as a result of the strong association between melittin and SDS, suggesting that the release of AMPs from BµE-based delivery vehicles may be hindered.

Mechanical formation of micro- and nano-plastic materials for environmental studies in agricultural ecosystems.

Release of microplastics (MPs) and nanoplastics (NPs) into agricultural fields is of great concern due to their reported ecotoxicity to organisms that provide beneficial service to the soil such as earthworms, and the potential ability of MPs and NPs to enter the food chain. Most fundamental studies of the fate and transport of plastic particulates in terrestrial environments employ idealized MP materials as models, such as monodisperse polystyrene spheres. In contrast, plastics that reside in agricultural soils consist of polydisperse fragments resulting from degraded films employed in agriculture. There exists a need for more representative materials in fundamental studies of the fate, transport, and ecotoxicity of MPs and NPs in soil ecosystems. The objective of this study was therefore to develop a procedure to produce MPs and NPs from agricultural plastics (a mulch film prepared biodegradable polymer polybutyrate adipate-co-terephthalate (PBAT) and low-density PE [LDPE]), and to characterize the resultant materials. Soaking of PBAT films under cryogenic conditions promoted embrittlement, similar to what occurs through environmental weathering. LDPE and cryogenically-treated PBAT underwent mechanical milling followed by sieve fractionation into MP fractions of 840 µm, 250 µm, 106 µm, and 45 µm. The 106 µm fraction was subjected to wet grinding to produce NPs of average particle size 366.0 nm and 389.4 nm for PBAT and LDPE, respectively. A two-parameter Weibull model described the MPs' particle size distributions, while NPs possessed bimodal distributions. Size reduction did not produce any changes in the chemical properties of the plastics, except for slight depolymerization and an increase of crystallinity resulting from cryogenic treatment. This study suggests that MPs form from cutting and high-impact mechanical degradation as would occur during the tillage into soil, and that NPs form from the MP fragments in regions of relative weakness that possess lower molecular weight polymers and crystallinity.

Mechanism of protein extraction from the solid state by water-in-oil microemulsions.

The extraction of solid-phase alpha-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid alpha-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process.

Formation of polyol-fatty acid esters by lipases in reverse micellar media.

The synthesis of polyol-fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2-monoglyceride, and sugars and their derivatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50-60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl-enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of partitioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of "mixed" glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2-monoglyceride to 1-monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no conversion.

1-Monoglyceride production from lipase-catalyzed esterification of glycerol and fatty acid in reverse micelles.

Glycerol-fatty acid esterification has been conducted with lipase from R. delemar in water/AOT/isooctane reverse micellar media, with the major product being 1-monoglyceride, a useful food-emulsifier. 1,3-diglyceride was also synthesized, but to a much lesser extent. For a given set of initial conditions, the reaction productivity, measured in terms of the initial product formation rate, V(0), and the final or equilibrium concentration of product, is optimal for a particular concentration of each surfactant, fatty acid, glycerol, and water. Many of these optimal values correlate well with a "critical" region on the phase diagram. Also, results indicate lipase-catalyzed esterification stops due to the achievement of kinetic equilibrium expect for a few cases where enzyme deactivation is severe. Dynamic light scattering was employed to examine the influence of water, glycerol, and fatty acid on micellar and interfacial structure. Results from this technique indicate enzyme kinetic are linked to interfacial phenomena and the presence of substrates at the interfacial region.

Esterification reactions of lipase in reverse micelles.

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.