Phosphate is well-known for its ability to covalently bond to metal oxides,27,28 giving the potential to significantly alter their surface properties. Figure 5 shows that even at relatively low concentrations of PO4 3− , the ζ-potential of nano-CuO switches from positive to negative and becomes increasingly negative with increasing PO4 3− concentrations. This change in ζ-potential is likely due to the formation of a negatively charged layer of copper phosphate on the surface of the ENMs resulting in enhanced electrostatic repulsion, leading to the effects shown in Figure 4. The nonlinear effect on sedimentation seen at higher PO4 3− concentrations may be due to covalent bridging between phosphate-coated particles. While the addition of PO4 3− slightly increases the pH of the suspension , its effect on ζ potential is greater than what is predicted for the small change in pH observed . For example, Figure S2 predicts a ζ potential of 0 at pH 6, but the addition of 2.0 mgL−1 PO4 3− results in a decrease in the ζ potential from +7.79 to −12.2 mV at the same pH. Figure S2 also shows the relationship between pH and ζ potential for nano-Cu and Cu2 and may explain why the presence of PO4 3− did not have a large impact on their behavior in the waters tested. The addition of even low concentrations of PO4 3− to nano-CuO suspensions resulted in the polarity of the particle surface charges switching from positive to negative,macetas redondas grandes but both nano-Cu and Cu2 are negatively charged throughout the whole range of pH tested.
Consequently, any sorption of PO4 3− on the surfaces of these particles is not likely to influence their behavior. 3.3. Dissolution. The total amount of aqueous phase copper and the fraction of ionic copper in Cuaq for the three ENMs varied with pH and TOC, as hypothesized, but also with ionic strength. Cuaq was generally stable over the course of the experiment but tended to increase after ∼60 days, indicating that some long-term dissolution may have been occurring. For Cu2 and nano-CuO in hydroponic media , dissolution to ionic Cu is significant even in the presence of complex-forming ions. In freshwater , the presence of NOM reduces Cuaq and Cudis, possibly through a combination of chelation and by coating particle surfaces. A further decrease in Cudis is seen in storm runoff, which at 6.49 mg C L−1 has the highest TOC content of the waters tested .The rhizosphere is the area around the plant root in soil where microorganisms are densely populated and dynamic interactions between the plants and microorganisms occur. These complex interactions and the assemblage of microorganisms are established, shaped, and maintained by the plant root. Plants exude photosynthetically fixed carbons as nutrient sources, secondary metabolites, and signaling molecules to populate root ecological niches with beneficial microorganisms. This “rhizosphere effect” has important implications for plant growth and protections against biotic and abiotic stressors, and for geochemical carbon cycling in soil environments.
With the expected rise of severe, climate-change-related weather conditions such as drought and the growing need to increase plant productivity with sustainable agricultural practices, it is vital to gain a mechanistic understanding of the rhizosphere effect and engineer the rhizosphere to improve plant growth and resilience. To understand the mechanisms of the highly dynamic processes occurring in the rhizosphere, in situ interrogation of the system with high spatiotemporal resolution is necessary. However, due to the underground nature of the root system and the sheer complexity of the microbiome in the highly heterogeneous soil environment, studying root andmicrobial interactions has been challenging. For example, in a typical rhizosphere analysis experiment, the plant is uprooted from soil in the field or a pot at a defined time point, and the microbiome and other relevant chemicals are sampled in a destructive manner. This practice of uprooting the plant is limiting because the spatial information on microbial community members is lost and the same plant generally cannot be sampled over time. Further, there are many confounding variables, such as chemical reactions with minerals in soil that complicate the analysis and make the studies less reproducible and relevant across different locations and environments. To overcome these experimental hurdles, researchers have designed various types of specialized devices such as rhizotrons and microfluidics devices to improve specific aspects of sampling, analytics, and manipulation of the rhizosphere system, albeit often deviating substantially from the natural system.
Fluorescent microscopy is a promising tool for noninvasive in situ imaging of the microbial and root interactions with high spatiotemporal resolution. Developing in situ rhizosphere imaging methods is made especially more relevant as the microbial community colonization of the root and the persistence and succession patterns are likely dynamic and dependent on the developmental stage of the plant. Better knowledge in this can help guide synthetic microbial community inoculation protocols to optimize plant productivity. A notable development of in situ imaging devices is a microfluidic root chip by Massalha et al., in which real-time imaging of the microbial colonization of Arabidopsis thaliana seedlings was captured by high-resolution fluorescent confocal microscopy. The co-inoculation by the fluorescent protein expressing Bacillus subtilis and Escherichia coli showed preferential localization, where B. subtilis established more densely at the root tip while E. coli dispersed homogeneously around the root . While microfluidic devices have shown great promises in rhizosphere research, the micro-scale dimension of the root chamber limits the samples to be young seedlings of the model plants. Rhizotrons can support the growing plants in a chamber for a much longer period of time, but are not designed for high-resolution microscopy. Recently, we described the development of fabricated ecosystems that can support the various model plants for 3–4 weeks by having the bigger root chamber, which can be modified for conventional microscopy by using a glass slide as the lower plate.
We demonstrated that the EcoFAB can generate reproducible plant physiology and exudation phenotypes by conducting the multilab experiments with the model grass B. distachyon. While the EcoFAB gives much improved imaging capability over rhizotrons and the imaging of the plant root phenotypes such as root hair length and branching pattern have become much more accessible, imaging the microbial interactions with the root remains challenging. One reason is that the current design of the EcoFAB is not suitable for imaging with high-magnification and numerical aperture objectives to interrogate the micro-scale interactions at high resolution. Another reason is that the root can easily go in and out of the focal plane with the extra chamber height, a problem that would be exacerbated by using high-magnification objectives with a thinner focal plane. To overcome these imaging constraints,maceta 25l we modified our EcoFAB to be an imaging specific device that combined a larger root chamber to support longer growth times and the ability to image root and microbial interactions. By implementing the pillar structures in the root chamber, the effective chamber height for the root is reduced so that the root remains closer to the glass surface while maintaining the flow of media throughout the chamber. The polydimethylsiloxane device body is bonded with a thin cover glass to reduce the working distance between the sample and microscope objective. Consistent hydration and supplementation of nutrients become more challenging with the more mature plants transpiring at a greater rate and the thinner chamber profile reducing the volume. We modified the commercially available growth chamber with a watering port so that the Imaging EcoFAB is housed in a sterile environment with easy sterile additions of water or media . Imaging EcoFAB is designed to improve the imaging capability by reducing the effective height of the root chamber so that the plant root grows closer to the cover glass. By adding the pillar structures with dimensions of 1.5 mm × 1.5 mm × 2 mm and pillars separated by 300 µm from each other throughout the top of the root chamber , the effective chamber height became 1 mm and the B. distachyon root was forced to grow closer to the cover glass . The pillar structures allowed effective exchange of nutrients and chemicals across the root chamber and yielded plants with similar growth patterns to the original EcoFAB.
The dimensions of the pillars and the distance between them can be modified based on the plant type and experimental specifications. However, the 3D printers have limitations with the aspect ratio of the 3D structures and resolution of the features. The printability of the pillars needs to be experimentally tested for each type of printer and resin . Once the mold is printed and treated for PDMS casting , the PDMS body is bonded with the thin cover glass to reduce the distance between the objective and the root to accommodate high magnification objectives with the short working distance . The ability to spectrally distinguish colocalizing bacteria in the rhizosphere is benefi- cial to interrogate the microbial interactions, especially while imaging at high magnification and resolution to allow segmentation and cell counting for quantitative data analysis. To demonstrate this in an Imaging EcoFAB, P. simiae strains engineered to constitutively express the fluorescent proteins mTagBFP, mTurquoise2, EGFP, mVenus, mKO, mApple, mCherry, mKate2, and mCardinal were inoculated into a week-old sterile B. distachyon. After 3 days, the spectral images at 10×, 20×, and 40× magnification were acquired in the lambda mode of ZEN software as well as the linear unmixing of the spectral images . Due to the excitation and emission spectral overlaps, all 9 fluorescent proteins could not be distinctly visualized. We also observed that the blue emitting proteins mTagBFP and mTurquoise2 did not have detectable fluorescence signals, possibly due to slower growth due to greater metabolic burdens to express these proteins. We speculate that this may be due to the greater metabolic burdens to express these proteins. The spectral images analyzed using K-means clustering algorithm and the fluorescence signals were clustered into four spectral categories : green , orange , red , and background . These three colors are represented in the spectrally unmixed images from the lambda mode . In the 40× image, which is focused around root hairs , the individual bacterial strains are resolved at various densities . Using this image, the segmentation analysis was conducted by first generating the binary image with the fluorescence intensity threshold and then applying the dilation and erosion operations with the circular structuring element to separate out the individual bacterium . This initial analysis yielded the counting of 478 cells in the image. However, to improve the accuracy of cell counting in the rhizosphere, the 3D multi-spectral image using z-stack is necessary as the different orientations of the rod-shaped P. simiae yield either rod or circular shapes on 2D profile. With the improved software package of the microscope system that supports multi-spectral 3D segmentation using machine learning algorithm , the quantitative analysis of the rhizosphere microbiome will become more accurate and mainstream. Imaging EcoFAB can be used for studying the bacterial persistence and succession patterns in the rhizosphere with the same live plant in situ over a 3- to 4-week duration. To examine this application, we conducted a succession study using two fluorescent strains of P. simiae. These two strains have identical genomes except for the fluorescent protein, allowing for the demonstration of a succession experiment in the device where a first one strain is established in the rhizosphere, followed by a second stain co-locating. First, mTagBFP expressing P. simiae was inoculated to the 2-week-old sterile B. distachyon at OD600 of 0.5. After three days, establishment of the mTagBFP strain was confirmed by fluorescent imaging . Then, the P. simiae with mCherry expression was inoculated to the same plant at OD600 of 0.1 and imaged after 2 days, showing the colocalization of mTagBFP and mCherry fluorescence around the root tip of B. distachyon .In this study, we improved the rhizosphere imaging ability of the original EcoFAB by adding pillar structures on the top of the root chamber and by using a thin cover glass as the bottom substrate. The device’s improvement was demonstrated by high-magnification and -resolution confocal imaging of the entire B. distachyon root and microbial interactions while maintaining the key advantages of the EcoFAB: real-time in situ imaging using conventional microscope system, bigger chamber to support the model plants for 3 to 4 weeks, ability to pack solid substrates, and improved environmental control such as sterility of the chamber.