Establishment of a transparent soil system to study Bacillus subtilis chemical ecology

Bacterial secondary metabolites are structurally diverse molecules that drive microbial interaction by altering growth, cell differentiation, and signaling. Bacillus subtilis, a Gram-positive soil-dwelling bacterium, produces a wealth of secondary metabolites, among them, lipopeptides have been vastly studied by their antimicrobial, antitumor, and surfactant activities. However, the natural functions of secondary metabolites in the lifestyles of the producing organism remain less explored under natural conditions, i.e. in soil. Here, we describe a hydrogel-based transparent soil system to investigate B. subtilis chemical ecology under controllable soil-like conditions. The transparent soil matrix allows the growth of B. subtilis and other isolates gnotobiotically and under nutrient-controlled conditions. Additionally, we show that transparent soil allows the detection of lipopeptides production and dynamics by HPLC-MS and MALDI-MS imaging, along with fluorescence imaging of 3-dimensional bacterial assemblages. We anticipate that this affordable and highly controllable system will promote bacterial chemical ecology research and help to elucidate microbial interactions driven by secondary metabolites.

Plant–environment microscopy tracks interactions of Bacillus subtilis with plant roots across the entire rhizosphere

Our understanding of plant–microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants’ large volume of influence. Here, we present the development of three-dimensional live microscopy for resolving plant–microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor-made, dual-illumination light sheet system acquired photons scattered from the plant while fluorescence emissions were simultaneously captured from transparent soil particles and labeled microorganisms, allowing the generation of quantitative data on samples ∼3,600 mm3 in size, with as good as 5 µm resolution at a rate of up to one scan every 30 min. The system tracked the movement of Bacillus subtilis populations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favored small pore spaces over the surface of soil particles, colonizing the root in a pulsatile manner. Migrations appeared to be directed toward the root cap, the point of “first contact,” before the subsequent colonization of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant–microbe interactions.

Hydrogeology Response to the Coordinated Mining of Coal and Uranium: A Transparent Physical Experiment

The migration of fracture and leaching solute caused by mining activity is critical to the hydrogeology. To characterize liquid and solid migration in a mining area of intergrown resources, the coordinated mining of coal and uranium was considered, and a physical experiment based on transparent soil was conducted. A well experimental performance of transparent soil composed of paraffin oil, n-tridecane, and silica gel and the leaching solution comprised of saturated oil red O dye was observed for hydrogeology characterization. An “arch-shaped” fracture zone with a maximum height of 90 m above the mined goaf and a “horizontal-shaped” fracture zone with a fractured depth of 9.97–16.09 m in the uranium-bearing layer were observed. The vertical leachate infiltration of 4.83 m was observed in the scenario of uranium mining prior to coal, which is smaller than those in the scenarios of comining of coal and uranium (10.26 m) and coal mining prior to uranium (16.09 m). A slight strata movement below the uranium was observed, and the leaching solution infiltration in the coal mining area was not observed in a short period in the scenario of uranium mining prior to coal; both of those was presented in the scenarios of comining of coal and uranium and coal mining prior to uranium.

In situ laser manipulation of root tissues in transparent soil

Aims Laser micromanipulation such as dissection or optical trapping enables remote physical modification of the activity of tissues, cells and organelles. To date, applications of laser manipulation to plant roots grown in soil have been limited. Here, we show laser manipulation can be applied in situ when plant roots are grown in transparent soil. Methods We have developed a Q-switched laser manipulation and imaging instrument to perform controlled dissection of roots and to study light-induced root growth responses. We performed a detailed characterisation of the properties of the cutting beams through the soil, studying dissection and optical ablation. Furthermore, we also studied the use of low light doses to control the root elongation rate of lettuce seedlings (Lactuca sativa) in air, agar, gel and transparent soil. Results We show that whilst soil inhomogeneities affect the thickness and circularity of the beam, those distortions are not inherently limiting. The ability to induce changes in root elongation or complete dissection of microscopic regions of the root is robust to substrate heterogeneity and microscopy set up and is maintained following the limited distortions induced by the transparent soil environment. Conclusions Our findings show that controlled in situ laser dissection of root tissues is possible with a simple and low-cost optical set-up. We also show that, in the absence of dissection, a reduced laser light power density can provide reversible control of root growth, achieving a precise “point and shoot” method for root manipulation.

Visualization of an internal sand displacement field around a laterally loaded vertical pile

In this research, an internal sand displacement field around a laterally loaded vertical pile is visualized using transparent soil and an image processing technique called digital image cross-correlation (DIC). DIC is a region-based image processing technique which can calculate the displacement field between two images. Transparent soil is made of silica gel with a pore fluid having the same refractive index. Transparent soil has been studied to have the strength and deformation properties similar to natural soil. An optical test set-up is developed to capture the images during loading. This optical test set-up consists of a camera, a laser light, a line generator lens, a loading frame, a Plexiglas mould, and a PC. The saturated fine sand in loose condition is modeled in this research. A laser light sheet is generated to slice the transparent soil model by passing a laser beam through the line generator lens. A distinctive laser speckle pattern is generated through the interaction between the laser light and transparent soil. A series of images are taken from the camera while a scaled pile is being loaded laterally. The displacement fields are calculated by cross-correlating two consecutive images and the corresponding strain fields are deduced from the displacement fields. The development of both displacement and strain fields is investigated by studying deformation and strains at different loading stages. The test results are similar to the published data. This research improves the understanding of soil movement around a laterally loaded pile. It also advances the physical modeling technique using transparent soil.