QELBY „-Induced Enhancement of Exclusion Zone Buildup and Seed Germination

A hydrophilic powder, QELBY, from the feldspar family of clay minerals was investigated for its ability to form structured or exclusion-zone (EZ) water. We demonstrated microsphere-free zones around different fractions of the QELBY powder or its hydrated pellet. Averaging approximately 100μm, these zones grew to a size similar to that formed in the vicinity of the Nafion standard. In the case of silica (control), only occasional microsphere-free zones of about 70μm were found. Further, studies to investigate QELBY’s energizing effect on germination and early sapling growth in brown chickpea seeds showed at least a 2-3-fold increase in root length and/or formation of shoots.This was seen in seeds bathed in QELBY supernatants or surrounded by QELBY powder outside the vials containing the seeds. This indirect effect was observed whether the QELBY was dry or hydrated.


Introduction
A highly hydrophilic material (QELBY) developed by the Quantum Energy Company is manufactured through a special process from the feldspar family of clay minerals.Details and composition are presented in a Korean patent (KP 10-1172018) [1] and published elsewhere [2].This hydrophilic material shows variety of interesting properties, mostly related to the enhancement of various cell-biological processes [3,4].Because of the diversity of enhancement effects, it is presumed that the mechanism is related to "something" present throughout the cell, the most likely substance being water.For example, one of the most important properties of QELBY is the absorption of ultraviolet light and emission of infrared light [2].According to the explanation of water structure proposed by Pollack [5], this infrared light can be a source of energy for the formation of structured water, otherwise known as exclusion zone (EZ) water.
In addition to this optical feature, QELBY powder shows very high zeta potential as well as semiconductive behavior [6].This implies high surface polarity, which can be a seeding point for EZ growth.Both characteristics of QELBY, a nucleation site for EZ growth, and an infrared energy-generating substance for the formation of EZ water, can be anticipated to support the enhanced formation of EZ around QELBY powder grains.
In this report we explored whether an EZ forms around QELBY powder grains and compared it to the EZ that is formed around other hydrophilic substances.We also investigated the dynamics of EZ growth.As a control we used natural silica powder, chosen because silica is the major component of QELBY.The study was also undertaken to complement earlier studies on cellular models of health and disease with promising results [3].Since EZs were observed around QELBY particles, we explored whether the water that was in contact with QELBY, when separated centrifugally from the powder, could still influence early plant growth, specifically seed germination and early sapling formation.Carboxylate-coated and polystyrene-coated microspheres are negatively charged, whereas the hollow glass spheres are electrically and chemically neutral and neutrally buoyant.The volume ratio of microsphere to water was 1 : 600 or 1 : 1000 depending on the kind of experiment performed and kept constant to eliminate any effects that might arise from concentration differences.

Setup.
Ordinary glass slides (Thermo Fisher Scientific) were used for examining pattern formation in QELBYand silica-containing water droplets positioned with a 1-ml latex-free syringe.For the microsphere exclusion studies, we used a specially formulated glass-slide chamber built from a rectangular polycarbonate plastic block (48 mm × 26 mm), with a hollow cylinder cut out (15 mm diameter, 4 mm deep) in the center, secured to an uncoated glass slide (Thermo Fisher Scientific) at the bottom (Figure 1).Some studies were conducted in the 8-well chambered slide (8-well Permanox slide; Lab-Tek).

Pattern Formation in Water
Droplets.A 0.1% (weight by volume) suspension of QELBY was prepared in DI water obtained from a Barnstead D3750 Nanopure Diamond water purification system (type 1 HPLC grade, 18.2 MΩ).The suspension was stirred for 2 hours in a commercial blender and a portion was filtered using Sharkskin General-Purpose filter paper (8-m to 12-m pore size; Whatman5) to remove the larger particles.A similar procedure was performed with a 0.1% suspension of silica powder in DI water.However, due to the small size of the particles (less than 8 to 10 m), the silica suspension was filtered with a 2.5 m pore sized, ash-less, Grade 42 Whatman quantitative filter paper.The filtered and unfiltered QELBY and silica water suspensions were examined using the "droplet evaporation method."

Exclusion of Microspheres in a Particle Suspension.
A few particles of QELBY or silica powder were carefully placed at the bottom of the 8-well chambered Permanox or the polycarbonate plastic chambered glass slide.The chamber was gently filled with 250 l of a diluted microsphere suspension (volume ratio of microsphere to DI water was 1 : 1000).Each type of microsphere listed above was tested.The interaction of microspheres with the particles was observed using an inverted microscope (Axio Observer A1, Zeiss).All image processing was done via ImageJ software.

Exclusion of Microspheres in Hydrated
Pellets.A 1% suspension of QELBY or silica powder (weight by volume) in DI water was vortexed overnight horizontally at room temperature.After centrifuging the mixture at 3000 rpm for 45 minutes, the supernatant was removed and labelled as QE-SUP or Si-SUP while the residue remaining behind was labelled as QE-RES or Si-RES, respectively.When compared with the white Si-RES, the QE-RES stacked as a tricolored residue (three hues of brown overlaid on one another).A few microliters of the well-mixed residue were pipetted to form tiny pellets (about 0.5-0.8mm) on the chambered slide.The pellets were allowed to dry at room temperature for at least one hour.
The chambered slide was placed on the stage of a Zeiss Axio Observer A1 inverted optical microscope and gently filled with 450 L of the diluted microsphere suspension (volume ratio of the polycarboxylate-coated 2 m microsphere to DI water was 1 : 600).The interaction of the microspheres with the QELBY (QE-RES) or silica (Si-RES) pellets was observed in the bright field mode with a 5x objective lens, which allowed visualization of the pellet in its entirety.Exclusion of microspheres in the vicinity of the two kinds of pellets was examined as a function of time.All image processing was done using ImageJ software.

Preparation of Powder Supernatants for UV-Visible Spectroscopy.
We placed a 1% suspension (weight by volume) of QELBY or silica powder in DI water contained in a 50 ml polypropylene tube.This was vortexed for 2 h or overnight at room temperature and centrifuged thereafter at 3000 rpm for 45 minutes to obtain the powder supernatant (QE-SUP or Si-SUP).These were scanned over a wavelength range of 200-400 nm using the Cary UV-Vis-NIR 5000 model as per the manual instructions.

Seed Germination and Sapling
Growth.Biological effects of the QELBY powder and their powder supernatants were examined via the "contact" and "noncontact" model experiments.

Contact Model.
In this model, we placed seeds in direct contact with 1-2 ml of the powder supernatants (QE-SUP) prepared as described above in Section 2.4.Both the 2 h and overnight-vortexed powder preparations were tested.For this study we selected regular brown chickpeas and those already "sprouted" under controlled conditions of light, temperature, and humidity.Controls were set up with seeds/sprouts immersed in similar volumes of DI water or Si-SUP.Two types of containers, 15 ml polypropylene, nonpyrogenic, high clarity tubes (Corning, Inc) as well as liquid scintillation glass vials (chemically inert, high clarity, from standard borosilicate glass), were examined for their ability to support seed germination and sapling growth under regular conditions of laboratory light and temperatures of 22-23 ∘ C. Seeds were checked for germination (protrusion of radicle) after 1-2 days, and saplings were harvested after one week.

Noncontact Model.
Seeds selected for similar appearance and weights were rinsed and placed in DI water in vials.These were, in turn, placed in plastic petri dishes that contained equal amounts of wet or dry QELBY (Figure 2).Controls consisted of the same arrangement except that the petri dishes contained water or equal amounts of wet or dry silica powder.Setups were separated by a rack of aluminum foil or placed 12-14 inches from each other and placed under standard conditions of laboratory light at temperatures of 22-23 ∘ C. Seeds were checked for protrusion of radicle (germination) after 1-2 days.Sprouted saplings were removed from the vials at the end of a one-week period, blotted gently with a soft paper towel, and weighed immediately.A few experiments were harvested after a one-month period.
2.6.Data Analysis.Seed germination is normally described as a physiological process that begins with water imbibition by seeds and culminates with the protrusion of the radicle.The definition may change according to the length of the radicle, ranging from 1 to 5 mm [7].In the current study, we recorded seeds that showed emergence of the radicle through its seed coat (testa) as being germinated."Percentage seed germination" was thus defined as the percentage of seeds with emerged radicles (1 mm) when compared to the total number of seeds used for experimentation.In some cases, we further substantiated these results by defining "percentage of saplings with shoots" as the percentage of germinated saplings that developed a shoot in addition to the root.

Pattern Formation in Water Droplets.
Although droplet evaporation studies generally focus on the patterns remaining after full evaporation, we explored the patterns occurring at various stages during the evaporation process, moving the microscope stage minutely in order to observe different regions.
As depicted in Figure 3, it was possible to observe clear zones of a few hundred microns around single particles in the evaporating droplets formed from the QELBY suspension.These clear zones persisted until the evaporation process was almost complete.Towards the end, a flow occurred, mixing all the particles together, leaving a typical "coffee-ring" pattern on the glass slide.However, even in the dried pattern it was still possible to recognize some clear areas (Figure 4).
Clear areas around single particles were distinctly observed in 21 of 30 droplets of QELBY-containing water thereby suggesting a fundamental role for the QELBY interaction with water.In the case of droplets containing silica powder, clear areas were observed in only two of the 30 droplets and their sizes were less than 100 m.A summary of results is shown in Figure 5.

QELBY Particles in
Microsphere Suspensions.Similar to that observed in the droplet-evaporation experiment, QELBY particles placed in a suspension of microspheres often generated microsphere-free zones (Figure 6).Microsphere exclusion was long lasting.We could observe clear zones for more than 24 hr.However, quantifying the fraction of particles generating exclusion was not easy because of the challenge of identifying individual particles.Some particles showed large microsphere-free zones, which resembled the particle-free zones described above (Figure 3(a)) while others failed to show any exclusion of microspheres at all.Instead, the microspheres would actually gather on the surface of some of the QELBY particles.This diversity of behaviors implied that different fractions of the QELBY powder might show distinctly different features (see Figure 9).
On the other hand, similar experiments with silica displayed only occasional microsphere-free zones; those that were observed were obviously smaller than those surrounding the QELBY particles (Figure 7).
The QELBY exclusion zones described above resembled those zones seen next to other hydrophilic surfaces such as Nafion.In fact, the exclusion zones in the vicinity of QELBY particles grew with a velocity comparable to that observed in the vicinity of Nafion (∼1 m/s) (Figure 8).

3.3.
Microsphere-Free Zones around Hydrated Pellets. Figure 9 shows representative images of the microsphere-free zones formed adjacent to the QE-RES and Si-RES pellets (formulated as described in Procedures), following a 20-minute exposure to microspheres.Capturing images every four minutes confirmed reasonably stabilized EZs.ImageJ software was used to measure the sizes of the microsphere-free zones.
Since the microsphere-free zones were typically nonuniform, some computation was necessary.We measured the area of the pellet (A) and that of the pellet plus microspherefree zone (A1).Computing the difference and dividing that difference by the pellet circumference gave the mean EZ width.We performed this calculation at four intervals over the 20-minute span and averaged the results.Experiments were repeated, each time with a different pellet sample.The mean EZ size for the QE-RES pellet was 107 ± 12 m versus 74 ± 9 m for the Si-RES pellet, which served as control.The  modest SD indicated that the EZ appeared to be reasonably stable over time.The results are summarized in Table 1.

UV-Visible Spectroscopy of Powder Supernatants.
When compared with the silica supernatants, the QELBY supernatants showed a broad bulge between 240 and 280 nm.Overall, powder suspensions vortexed overnight had higher absorbance units than those vortexed for 2 h only ( = 10).Representative figures, Figures 10(a

QE-RES pellet
Si-RES pellet Average EZ size (m) ± SD 107 ± 12 * 74 ± 9 * placed in direct contact with the different supernatants as described in the Experimental Procedures and observed for seed germination and sapling growth.Irrespective of the type of container ( = 4; each kind), seeds immersed in QE-SUP were the first to germinate ( = 12).At the end of a week, while there was no significant difference in weight, young saplings in QE-SUP were at least 3-4 times longer than those in an equal volume of DI water or Si-SUP (Figures 11(a) and 11(b)).
Sprouted Chickpea Seeds.Germination amongst a population of seeds is not synchronous.Hence we minimized the variability by immersing chickpea seeds that were already sprouted under controlled conditions, as described in Experimental Procedures.After selecting for similar weights and appearance, sprouted chickpea seeds were immersed in the powder supernatants for a week's growth in the two types of containers.Irrespective of whether the container was a tube or vial, we found no significant change in the weight of the seedlings.However, roots of sprouted chickpea seedlings were significantly longer when grown in QE-SUP (58%) versus Si-SUP and about 24% when compared with DI water (Figure 12).

Noncontact Model: Chickpea Seeds.
In these experiments, the surrounding powder was not in direct contact with the seed; the vial wall separated the two.As depicted in A few experiments were prolonged over a month-long period.Results presented in Figure 14 depict well-formed stems with abundant green leaves in chickpea saplings surrounded by wet/dry QE powder relative to either DI water or wet/dry Si powder (Figure 14;  = 3-6).

Discussion
The results confirm that QELBY powder builds EZ water and powerfully acts to enhance plant growth.These effects were borne out by several independent methods.
In a droplet-evaporation method we observed clear, microsphere-free zones surrounding individual QELBY particles.The zones were generally on the order of several hundred micrometers in width.Similarly, particles examined in aqueous microsphere suspensions generated microspherefree zones that lasted at least for the 24 hours of observation.Zone sizes were variable, reflecting the multicomponent nature of QELBY particles.
These microsphere-free zones resemble the ones seen next to various hydrophilic surfaces, which serve to indicate the presence of EZ water [5].In fact, typical EZ size was only slightly smaller than that seen next to Nafion, which has become a standard for studies of EZ development.Rate of growth was also comparable.
We also examined supernatants obtained from QELBY suspensions to determine whether EZ water was present.UV-VIS absorption spectra showed an absorption peak near 270 nm, which is indicative of the presence of EZ water [8].This finding provided additional indication that QELBY particles nucleate EZ growth and correlates with the substantial EZ-growth rate.
The reason why the supernatants are so effective in building EZ is not yet clear.Possibly some small mineral remnants present in the supernatants nucleate EZ buildup.Structurebuilding ions might do the same.Alternatively, small EZ fractions may survive centrifugation and wind up in the supernatant.Future studies may settle the issue.Given QELBY's capacity to nucleate EZ buildup, we explored whether exposure of QELBY to biological specimens might enhance natural biological processes.We exposed QELBY to regular and sprouted chickpea seeds and found that sapling growth was enhanced relative to controls.A similar trend was observed with radish seeds (data not shown).We speculate that QELBY was effective on the cells at the growing tip of the sapling roots and resulted in their elongation.Enhancement was seen not only when the seeds were bathed in centrifuged QELBY supernatants, but also when QELBY powder was placed immediately outside the vials containing the seeds.This indirect effect was observed whether the QELBY was dry or hydrated.
This action-at-a-distance effect reflects the growing awareness that information can be transmitted between samples of water by electromagnetic signals.When vials containing certain suspended or dissolved materials are placed next to vials of pure water, the former can alter the water's structure, as inferred from characteristic changes of infrared absorption spectrum [9].Further, vials of water placed near samples of DNA can be used to create new DNA with the same sequence as the original [10].While the results obtained here do not go as far as those studies, they do show that growth-enhancing effects can occur without direct contact.
In sum, QELBY material, originating from natural clay, has the capacity to nucleate EZ buildup.And possibly through the vehicle of EZ buildup, QELBY has an enhancing effect on plant growth, a feature with considerable practical value.

Figure 2 :Figure 3 :
Figure 2: Noncontact model: diagrammatic representation of brown chickpeas immersed in deionized water (DI) water contained in liquid scintillation vials.The vials were surrounded by one of several materials contained in petri dishes: DI water, dry/wet QE or Si.

Figure 4 :Figure 5 :
Figure 4: QELBY powder droplet evaporation viewed over time until the complete evaporation after 12 minutes.

Figure 6 :
Figure 6: Clear zones, free of microspheres, were generated around single QELBY particles in various chambers and microsphere types (a-d).

Figure 7 :
Figure 7: Clear zones, free of microspheres, were generated around single Silica particles in microsphere suspensions of various types, but smaller and rarer than around QELBY particles.

Figure 8 :
Figure 8: Rate of exclusion zone formation around QELBY particles versus next to Nafion.QELBY particles in polycarboxylate-coated 2-m aqueous suspension, and Nafion TT110 in polycarboxylatecoated 2-m microsphere suspension were placed separately in an 8-well chambered Permanox slide.The curves represent the means of three measurements.

Figure 9 :Figure 10 :
Figure 9: Representative images of exclusion zone development next to pellets of QE-RES (a) and Si-RES (b), also detailing the use of the Image J software to obtain area measurements.Double-sided arrows represent a few of the marked EZs.

Figure 12 :
Figure 11: Representative images of early germination and growth observed in chickpea seeds immersed in equal volumes of DI water, Si-SUP, or QE-SUP in (a) test tubes or (b) liquid scintillation vials.

Figure 13 :
Figure 13: Effect of dry and wet QE and Si powders on the (a) weight (mg) and (b and c) length (mm) of roots (R) and shoots (S) of chickpea saplings after one week (n = 12-18).

Table 2 :
Percentage seed germination and percentage of saplings with shoot growth in chickpea seeds surrounded by wet/dry powder during a one-week growth period (n: 8-18).