Scanning Electron Microscopy

Example 1

95 g of manganese (purity: 99.95%; purchased from Taewon Scientific Co., Ltd.) and 5 g of high-purity graphite (purity: 99.5%; purchased from Taewon Scientific Co., Ltd.) were placed in a water-cooled copper crucible of an argon plasma arc melting apparatus (manufactured by Labold AG, Germany, Model: vacuum arc melting furnace Model LK6/45), and melted at 2,000 K under an argon atmosphere. The melt was cooled to room temperature at a cooling rate of 10⁴ K/min to obtain an alloy ingot. The alloy ingot was crushed to a particle size of 1 mm or less by hand grinding. Thereafter, the obtained powders were magnetically separated using a Nd-based magnet to remove impurities repeatedly, and the Mn₄C magnetic powders were collected. The collected Mn₄C magnetic powders were subjected to X-ray diffraction (XRD) analysis (measurement system: D/MAX-2500 V/PO, Rigaku; measurement condition: Cu—K_α ray) and energy-dispersive X-ray spectroscopy (EDS) using FE-SEM (Field Emission Scanning Electron Microscope, MIRA3 LM).

FIGS. 2(a) and 2 (b) show an X-ray diffraction pattern and an energy-dispersive X-ray spectroscopy graph of the Mn₄C magnetic material produced according to Example 1 of the present disclosure, respectively.

As can be seen in FIG. 2(a), the Mn₄C magnetic material showed diffraction peaks of (111), (200), (220), (311) and (222) crystal planes at 2θ values of 40°, 48°, 69°, 82° and 88°, respectively, in the XRD analysis. Thus, it can be seen that the XRD patterns of the Mn₄C magnetic material produced according to Example 1 are well consistent with the patterns of the cubic perovskite Mn₄C. In addition, the Mn₄C magnetic material shows several very weak diffraction peaks that can correspond to Mn₂₃C₆ and Mn. That is, the diffraction peak intensity at 2θ values of 43° and 44°, which correspond to Mn and Mn₂₃C₆ impurities, is as very low as about 2.5% of the diffraction intensity of the peak corresponding to the (111) plane. Through this, it can be seen that the powders obtained in Example 1 have high-purity Mn₄C phase. The lattice parameter of the Mn₄C is estimated to be about 3.8682 Å.

FIG. 2(b) shows the results of analyzing the atomic ratio of Mn:C in the powder by EDS. The atomic ratio of Mn:C is 80.62:19.38, which is very close to 4:1 within the experimental uncertainties. Thus, it can be seen that the powder is also confirmed to be Mn₄C.

The M-T curve of the field aligned Mn₄C powder obtained in Example 1 was measured under an applied field of 4 T and at a temperature ranging from 50 K to 400 K. Meanwhile, the M-T curve of the randomly oriented Mn₄C powder was measured under an applied field of 1 T. The Curie temperature of Mn₄C was measured under 10 mT while decreasing temperature from 930 K at a rate of 20 K/min.

FIGS. 3(a) to 3(c) show the M-T curves of the Mn₄C magnetic material, produced according to Example 1 of the present disclosure, under magnetic fields of 4 T, 1 T, and 10 mT, respectively.

FIG. 3 shows magnetization-temperature (M-T) curves indicating the results of measuring the temperature-dependent magnetization intensity of the Mn₄C magnetic material, produced in Example 1, using the vibrating sample magnetometer (VSM) mode of Physical Property Measurement System (PPMS®) (Quantum Design Inc.).

According to the Néel theory, the ferrimagnets that contain nonequivalent substructures of magnetic ions may have a number of unusual forms of M-T curves below the Curie temperature, depending on the distribution of magnetic ions between the substructures and on the relative value of the molecular field coefficients. The anomalous M-T curves of Mn₄C, as shown in FIG. 3(a), can be explained to some extent by the Néel's P-type ferrimagnetism, which appears when the sublattice with smaller moment is thermally disturbed more easily. For Mn₄C with two sublattices of Mn_I and Mn_II, as shown in FIG. 1, the Mn_I sublattice might have smaller moment.

FIG. 3(a) shows the temperature dependence of magnetization of the Mn₄C magnetic material produced in Example 1. The magnetization of Mn₄C measured at 4.2K is 6.22 Am²/kg (4 T), corresponding to 0.258μ_B per unit cell. The magnetization of the Mn₄C magnetic material varies little at temperatures below 50 K, and is quite different from that of most magnetic materials, which undergo a magnetization deterioration with increasing temperature due to thermal agitation. Furthermore, the magnetization of the Mn₄C magnetic material increases linearly with increasing temperature at temperatures above 50 K. The linear fitting of the magnetization of Mn₄C at 4 T within the temperature range of 100 K to 400 K can be written as M=0.0072T+5.6788, where M and T are expressed in Am²/kg and K, respectively. Thus, the temperature coefficient of magnetization of Mn₄C is estimated to be about ˜2.99*10⁻⁴μ_B/K per unit cell. The mechanisms of the anomalous thermomagnetic behaviors of Mn₄C may be related to the magnetization competition of the two ferromagnetic sublattices (Mn_I and Mn_II) as shown in FIG. 1.

FIG. 3(b) shows the M-T curves of the Mn₄C powders at temperatures within the range of 300 K to 930 K under 1 T. The linear magnetization increment stops at 590 K, above which the magnetization of Mn₄C starts to decrease slowly first and then sharply at a temperature of about 860 K. The slow magnetization decrement at temperatures above 590 K is ascribed to the decomposition of Mn₄C, which is proved by further heat-treatment of Mn₄C as described below.

According to one embodiment of the present disclosure, the saturation magnetization of Mn₄C increases linearly with increasing temperature within the range of 50 K to 590 K and remains stable at temperatures below 50 K. The increases in anomalous magnetization of Mn₄C with increasing temperature can be considered in terms of the Néel's P-type ferrimagnetism. At temperatures above 590 K, the Mn₄C decomposes into Mn₂₃C₆ and Mn, which are partially oxidized into the manganosite when exposed to air. The remanent magnetization of Mn₄C varies little with temperature. The Curie temperature of Mn₄C is about 870 K. The positive temperature coefficient (about 0.0072 Am²/kgK) of magnetization in Mn₄C is potentially important in controlling the thermodynamics of magnetization in magnetic materials.

The Curie temperature T_e of Mn₄C is measured to be about 870 K, as shown in FIG. 3(c). Therefore, the sharp magnetization decrement of Mn₄C at temperatures above 860 K is ascribed to both the decomposition of Mn₄C and the temperature near the Tc of Mn₄C.

FIG. 4 is a graph showing the magnetic hysteresis loops of the Mn₄C magnetic material, produced according to Example 1 of the present disclosure, at 4.2 K, 200 K and 400 K. The magnetic hysteresis loops were measured by using the PPMS system (Quantum Design) under a magnetic field of 7 T while the temperature was changed from 4 K to 400 K.

As shown in FIG. 4, the positive temperature coefficient of magnetization was further proved by the magnetic hysteresis loops of Mn₄C as shown in FIG. 4. The Mn₄C shows a much higher magnetization at 400 K than that at 4.2 K. Moreover, the remanent magnetization of Mn₄C varies little with temperature and is Δ3.5 Am²/kg within the temperature range of 4.2 K to 400 K. The constant remanent magnetization of Mn₄C within a wide temperature range indicates the high stability of magnetization against thermal agitation. The coercivities of Mn₄C at 4.2 K, 200 K, and 400 K were 75 mT, 43 mT, and 33 mT, respectively.

The magnetic properties of Mn₄C measured are different from the previous theoretical results. A corner Mn_I moment of 3.85μ_B antiparallel to three face-centered Mn_II moments of 1.23μ_B in Mn₄C was expected at 77 K. The net moment per unit cell was estimated to be 0.16μ_B. In the above experiment, the net moment in pure Mn₄C at 77 K is 0.26μ_B/unit cell, which is much larger than that expected by Takei et al. It was reported that the total magnetic moment of Mn₄C was calculated to be about 1μ_B, which is almost four times larger than the 0.258μ_B per unit cell measured at 4.2 K, as shown in FIG. 4.

FIG. 5 is an enlarged view of the temperature-dependent XRD patterns of the Mn₄C magnetic material produced according to Example 1 of the present disclosure.

The thermomagnetic behaviors of Mn₄C are related to the variation in the lattice parameters of Mn₄C with temperature. It is known that the distance of near-neighbor manganese atoms plays an important role in the antiferro- or ferro-magnetic configurations of Mn atoms. Ferromagnetic coupling of Mn atoms is possible only when the Mn—Mn distance is large enough. FIG. 5 shows the diffraction peaks of the (111) and (200) planes of Mn₄C at temperatures from 16 K to 300 K. With increasing temperature, both (111) and (200) peaks of Mn₄C shifted to a lower degree at temperatures between 50 K and 300 K, indicating an enlarged distance of Mn—Mn atoms in Mn₄C. No peak shift is obviously observed for Mn₄C at temperatures below 50 K. The distance of nearest-neighbor manganese atoms plays an important role in the antiferro- or ferro-magnetic configurations of Mn atoms and thus has a large effect on the magnetic properties of the compounds.

Thus, it can be seen that the abnormal increase in magnetization of Mn₄C with increasing temperature occurs due to the variation in the lattice parameters of Mn₄C with temperature.

The powder produced in Example 1 was annealed in vacuum for 1 hour at each of 700 K and 923 K, and then subjected to X-ray spectroscopy, and the results thereof are shown in FIG. 6.

The magnetization reduction of Mn₄C at temperatures above 590 K is ascribed to the decomposition of Mn₄C, which is proved by the XRD patterns of the powders after annealing Mn₄C at elevated temperatures. FIG. 6 shows the structural evolution of Mn₄C at elevated temperatures. When Mn₄C is annealed at 700 K, a small fraction of Mn₄C decomposes into a small amount of Mn₂₃C₆ and Mn. The presence of manganosite is ascribed to the spontaneous oxidation of the Mn precipitated from Mn₄C when exposed to air after annealing. The fraction of Mn₂₃C₆ was enhanced significantly for Mn₄C annealed at 923 K, as shown in FIG. 6.

These results prove that the metastable Mn₄C decomposes into stable Mn₂₃C₆ at temperatures above 590 K. The presence of Mn₄C in the powder annealed at 923 K indicates a limited decomposition rate of Mn₄C, from which the Tc of Mn₄C can be measured. Both Mn₂₃C₆ and Mn are weak paramagnets at ambient temperature and elevated temperatures. Therefore, the magnetic transition of the Mn₄C magnetic material at 870 K is ascribed to the Curie point of the ferrimagnetic Mn₄C.

The Mn₄C shows a constant magnetization of 0.258μ_B per unit cell below 50 K and a linear increment of magnetization with increasing temperature within the range of 50 K to 590 K, above which Mn₂₃C₆ precipitates from Mn₄C. The anomalous M-T curves of Mn₄C can be considered in terms of the Néel's P-type ferrimagnetism.

Example 1

119 Dicty strains were screened for their ability to feed on Dickeya (Dd) or Pectobacterium (Pcc) at 10° C. This assay was performed by inoculating Dd or Pcc on a low nutrient medium (SM2 agar) that supports both bacterial and Dicty growth. Dicty spores from individual strains were then inoculated on top of the bacterial growth and incubated at 10° C. to mimic potato storage temperatures. Dicty strains that successfully fed on Dd or Pcc created visible clearings in the lawn of bacterial growth and ultimately produced sporangia (fruiting bodies) that rose from the agar surface. An example of the phenotype that was considered successful clearing of bacteria is shown in FIG. 3A. From this initial screen, 36 Dicty strains that were capable of feeding on both Dd and Pcc at 10° C. were identified (FIG. 1B).

Of the 36 strains capable of feeding on both Dd and Pcc, 34 came from the Group 4 Dictyostelids (FIG. 1). This group includes D. discoideum, D. giganteum, D. minutum, D. mucoroides, D. purpureum, and D. sphaerocephalum (72). The results indicate that this group is particularly enriched in Dd and Pcc-feeding strains.

A further experiment was performed to identify Dicty species capable of feeding on biofilms of Dd and Pcc. Microporous polycarbonate membranes (MPMs) are widely reported to support biofilm formation of numerous Enterobacteriaceae species (2, 63, 70, 71). It was determined if Dd and Pcc formed biofilms on MPMs and determined if Dicty strains were capable of feeding on these biofilms. Membranes were placed on top of SM2 agar to provide Dd and Pcc with nutrients for growth. Bacteria were then inoculated on the surface of the MPMs and growth was monitored over the course of 1 week by washing bacteria off the membranes and performing dilution plating for colony counting. Growth of both bacterial strains plateaued around 4 dpi (FIG. 2).

From these results, it was determined that the best time to collect inoculated MPMs for biofilm analysis was at 2 dpi. Scanning electron microscopy (SEM) is commonly used to confirm biofilm formation by detecting extracellular polymeric substance (EPS) that forms the biofilm matrix (2). Samples of Dd and Pcc after 2 days of growth on MPMs in the presence and absence of Dicty are analyzed using SEM.

19 Dicty strains identified as active were tested for their ability to feed on Dd and Pcc growing on MPMs. These experiments were performed by establishing Dd and Pcc growth on MPMs overlaid on SM2 agar at 37° C. for 24 hr. Dicty spores were then applied to the center of bacterial growth in a 5 uL drop containing 1000 spores. Bacteria and Dicty were incubated at 10° C. for 2 weeks before remaining bacteria were washed off and colonies were counted. Representative images of Dicty growing on Dd and Pcc on MPMs are shown in FIG. 3A.

No Dicty strains produced a statistically significant reduction in Dd viability compared to the non-treated control. However, treating Dd lawns with Cohen 36, Cohen 9, WS-15, WS-20, and WS-69 consistently reduced the number of viable bacteria by approximately 100,000-fold compared to the non-treated control (FIG. 3B). Cohen 9 was the only Dicty strain that produced a statistically significant reduction in viability of Pcc compared to the non-treated control (FIG. 3C). Other Dicty strains capable of reducing the number of viable Pcc by at least 100,000-fold were Cohen 35, Cohen 36, WS-647, and WS-69 (FIG. 3C).

It was observed that Dicty strains Cohen 9, Cohen 36, and WS-69 were capable of feeding on both Dd and Pcc when these bacteria were cultured on SM2 agar and MPMs (FIGS. 1 and 3). These strains were also particularly effective feeders as all three reduced the number of viable Dd and Pcc on MPMs at 10° C. by 100,000-fold compared to the non-treated control (FIGS. 3B and 3C).

To determine if these strains could suppress soft rot development on seed potato tubers, tubers were tab-inoculated with Dd or Pcc and treated with spores from each Dicty strain. Seed potatoes were surface-sterilized and punctured using a sterile screw to a depth of 1.5 mm. Overnight cultures of Dd and Pcc were suspended in 10 mM potassium phosphate buffer, diluted to an OD600 of approximately 0.003, and administered as a 5 μL drop into the wound. Next, 5 of a Dicty spore suspension (100,000 spores) was added to the wound. Inoculated seed potatoes were placed in a plastic container with moist paper towels and were misted with water twice a day to maintain a high humidity. After 3 days at room temperature, seed potatoes were sliced in half and the area of macerated tissue was quantified using ImageJ.

All three strains reduced the severity of soft rot caused by Dd and Pcc (FIG. 4). Cohen 36 was the most effective strain on both Dd and Pcc: reducing the area of tissue maceration by 60% and 35%, respectively (FIG. 4B). Treating seed potatoes with WS-69 reduced the area of tissue maceration by 50% and 30% for Dd and Pcc, respectively (FIG. 4B). Finally, Cohen 9 was the least effective, but still able to reduce tissue maceration caused by Dd and Pcc by 25% and 20%, respectively (FIG. 4B).

FIG. 7 shows that three Dicty isolates control Dd and Pcc in seed tubers (at 25° C.). Two sets of data from different weeks were normalized to the Dickeya or Pectobacterium only bacterial control. The average area of macerated potato tissue measured in mm² was set as “1” or “100%”. The average of all the other treatments including Dicty were divided by bacteria only control and multiplied by 100 to obtain a percentage. Each set contained 5 tubers per treatment.

Dicty should be capable of sporulating at temperatures as cold as 10° C. on a potato surface if they are applied as a one-time pre-planting or post-harvest treatment. Sporulation was assessed by inoculating small potato discs (5×6 mm) with 10 μL of Dd or Pcc suspensions at an OD600 of 3×10⁻⁵ and Dicty spores at a concentration of 1×10⁷ spores/mL. Potato discs were kept in a covered 96-well plate for two weeks at 10° C. followed by visual inspection for son using a dissecting microscope. Representative images of a strain producing many sori (WS-517) and a strain producing few sori (WS-69) are shown in FIG. 5. Of the 11 strains evaluated, only Cohen 9 and WS-20 were unable to sporulate in the presence of both pathogens (Table 1).

Example 2

This example describes the use of a high throughput screening assay to identify Dicty strains from Alaska (e.g., BAC10A, BAF6A, BAC3A, NW2, KB4A (ATCC® MYA-4262™) SO8B, SO3A, BAF9B, IC2A (ATCC® MYA-4259™), AK1A1 (ATCC® MYA-4272™) PBF4B (ATCC® MYA-4263), PBF8B, BSB1A, SO5B (ATCC® MYA-4249), PBF3C, PBF6B, NW2B, NW10B (ATCC® MYA-4271™), PBF9A, IC5A (ATCC® MYA-4256TH), ABC8A (ATCC® MYA-4260), NW16B, ABC10B, ABB6B (ATCC® MYA-4261), BA4A (ATCC® MYA-4252), AKK5A, AKK52C, HP4 (ATCC® MYA-4286), HP8 (ATCC® MYA-4284), or NW9A) that feed on Dd and Pcc at 10° C. on potatoes.

Results from 11 Dicty strains screened against Dd at 10° C. are presented in FIG. 6. Data was analyzed for significance using a one-way analysis of variance (ANOVA; alpha =0.05) with Tukey's honest significant difference (HSD) test to compare means between the treatments and the No Dicty control. A reduction in Dd proliferation when potato discs were treated with Dicty strains Cohen 9, Cohen 36, WS-15, Maryland 18a, BAF6A, NW2, and SO3A.

The Alaskan Dicty strains, and those identified in Example 1, are further tested against coinfections of Dd and Pcc. It is useful to identify Dicty strains that can suppress Dd and Pcc coinfections as these two pathogens have been isolated together from diseased potatoes (15). The ability of Dicty strains with different feeding preferences (Dd vs. Pcc) to complement each other when administered as a cotreatment is assayed.

Example 4

1 gram of PVDF was dissolved in 42.5 grams of acetone and 19 g of sodium chloride was added to form a solvent mixture by mixing at ambient temperature (about 23° C.). Portions of the solvent mixture were injected into 25 milliliters of water by rapidly squirting the solvent mixture into the water via a pipette and filtering the precipitate until all of the solvent mixture was precipitated. The PVDF fibrillated in the presence of the salt crystals and formed the composition. The filtering was performed by pouring over a coffee filter. The salt was washed out to result in a fibrillated PVDF that was free of the salt. The fibrillated PVDF was dried at 90° C. to remove acetone and water and the fibrillated PDVF was compressed to form a free-standing mat. The mat was then dried again at 130° C. The same process was performed without the presence of the salt and the PVDF precipitated from solution without forming the fibrils.

FIG. 7A is a scanning electron microscopy image of the PVDF after removal of the salt. Elemental analysis of the PDVF after the salt removal was performed and the results are shown in FIG. 7B and in FIG. 7C.