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Microbes govern most soil functions, but investigation of these processes at the scale of their cells has been difficult to accomplish. Here we incubate microfabricated, transparent ‘soil chips’ with soil, or bury them directly in the field. Both soil microbes and minerals enter the chips, which enables us to investigate diverse community interdependences, such as inter-kingdom and food-web interactions, and feedbacks between microbes and the pore space microstructures. The presence of hyphae (‘fungal highways’) strongly and frequently increases the dispersal range and abundance of water-dwelling organisms such as bacteria and protists across air pockets. Physical forces such as water movements, but also organisms and especially fungi form new microhabitats by altering the pore space architecture and distribution of soil minerals in the chip. We show that soil chips hold a large potential for studying in-situ microbial interactions and soil functions, and to interconnect field microbial ecology with laboratory experiments., Mafla-Endara et al. incubate a microfluidic chip with and directly in soil in order to examine interactions between microbial communities and the pore space microstructures. This work shows the spatiotemporal changes of soil microhabitats and demonstrates that fungal hyphae increase the dispersal range and abundance of water-dwelling organisms across air pockets.

Terrestrial ecosystems are under threat due to the continuous accumulation of plastics in soils. Particularly, microplastics have been proven to negatively affect the performance of soil macrofauna such as earthworms, as well as soil mesofauna including springtails and nematodes. Unfortunately, two big groups remain largely unexplored: the soil microfauna and microflora.Recent studies have shown that soil microbial community composition can significantly vary depending on the concentration and type of plastic, favouring some groups and disfavouring others. To have a better understanding of these relationships, it is necessary to study them at relevant scale: the microscale.Considering that in situ observations are hard to achieve due to the opacity of soil and ever-changing soil architecture, we used transparent micro-engineered chips to study interactions between microplastics and soil microorganisms live. We hypothesized that different concentrations of microplastics interfere with a natural microbial community in terms of 1. Soil microbial colonization/succession of the chips and 2. Soil microbial growth inside the chips’ pore space.We fabricated chips containing different microstructures that simulate soil pore spaces. The chips were bonded to a glass slide and one side was opened to allow microbial colonization. Each chip was filled with a mix of liquid nutrient medium and 1.0 µm polystyrene microbeads at microplastic concentrations of 0.0, 0.006, 0.001 and 0.0005 mg/ml. The chip´s opening was inoculated with 5 g of soil and incubated in the laboratory at room temperature for one month. We documented the presence/absence and abundance of different soil microbial groups changing over time by using an inverted microscope.Our preliminary study reveals that larger microorganisms are sensitive to the presence of microbeads 1.0 µm size. We found that all major soil microbial groups (fungi, bacteria, and protists) and nematodes colonized the chips. However, their abundance was affected by the presence of microplastics, irrespective of the concentration. Particularly protists and nematodes were lower in number during the first days of the exposure. The beads were clearly visibly taken up into the cells of the protists or the digestive tract of the nematodes.We are now investigating what consequences the lower abundance of certain soil microbial groups have for the soil food web. As seen here, micro-engineered chips are useful tools to provide visual access at the scale where most cell-to-cell interactions occur.

The effect of habitat complexity on microbial processes The way microbes behave in nature can vary widely depending on the spatial characteristics they are located in. This aspect of the microbial environment can determine whether processes such as organic matter degradation, nitrogen fixation, or microbial speciation, among others, occur and the extent to which they occur. Investigating how the different spatial characteristics of microhabitats influence microbes has been challenging mainly due to methodological limitations. In the case of soil sciences, attempts to describe the inner structure of the soil pore space, and to connect it to microbial processes, has been one of the main goals of the field in the last years. A major challenge in soil microbial ecology is to reveal the mechanisms that prevent nutrient limited soil microorganisms to access the soil organic matter pools. My project is directed towards answering the question of how spatial complexity affects microbial growth, and how this can lead to organic matter stabilization.Using microfluidic devices that were designed to mimic the inner soil pore physical complexity, we followed the effect of an increasing complexity in the growth and substrate degradation of bacterial and fungal lab strains. The parameters we used to measure complexity were two: the turning angle and order of pore channels, and the fractal order of a pore maze. When we tested the effect of an increasing in turning angle sharpness on microbial growth, we found that that as angles became sharper, bacterial and fungal growth decreased, but fungi were more affected than bacteria. We also found that the substrate degradation was only affected when bacteria and fungi grew together, being lower as the angles were sharper. This confirms the hypothesis that an increasing angle sharpness in an elongated pore space would decrease organic matter degradation. Our next series of experiments, testing the effect of maze fractal complexity, however, showed a different picture. While the effect of complexity on fungi was negative, similar to the previous experiments, bacteria were positively affected by maze complexity, growing more as mazes increased fractal iterations. Substrate degradation was also higher as mazes were more complex. In this case, the results were contrary to our hypothesis, especially for bacteria. To see the relevance of our results in natural microbial communities, we repeated both experiments on a soil microbial extract (containing mainly bacteria) and followed the substrate degradation patterns over time. We found, in this case, that as complexity increased, both in terms of angle sharpness and fractal order, the substrate consumption also increased. Our results show that the spatial complexity provides microbes an environment for a wide variety of ecological interactions to occur that lead to a higher substrate degradation efficiency.

Soils are characterized by their largely varying microhabitats that determine their microbial communities and functions such as nutrient cycling. Microbes, and especially fungi, do not only react to those microhabitats but also contribute to shaping them.We used transparent, microstructured chips simulating the internal pore space of soils, to microscopically study fungal mycelia at the hyphal scale. We investigated the variety of fungal morphologies in maze structures, and hyphal interactions with their biotic and abiotic microenvironments. We studied both a variety of laboratory strains including an arbuscular mycorrhizal fungus and natural soil inocula, and we quantified their growth strategies in different microstructures and their interactions with bacteria, protists and soil mineral particles.We could observe how the rigid hyphae of fungi opened up passages through chip- or soil solids and aggregates, increasing the spatial availability of the pore space. They, on the other hand, also filled up pores and pore necks with their biomass, creatingbarriers for both organisms, flow of water and sedimentation of matter, and thus changing the pore size distribution and -connectivity. Hyphae also increased the wettability of pores, which led to a higher connectivity of water films across air pockets and thus benefiting the dispersal of water-dwelling microorganisms, a phenomenon earlier termed “fungal highways”. We found the abundance of both bacteria and protists strongly increased in pore spaces containing hyphae in comparison to those without, dispersal events via fungal hyphae that happened frequently and were quantifiable in the high internal replication of our chip’s pore space channels. This allowed us to conclude on a high relevance of this mode of dispersal in soils with intermediate moisture. Fungal hyphae had thus a strong and obvious effect on their surrounding microenvironments and organisms.We consider the study of microbial behaviour and interactions at the cellular scale in microhabitats to be essential for a better understanding of soil functions, and to gain mechanistic insight into phenomena observable at macroscale.

Many soil processes are governed by microbes, and biological and physical processes influence each other. We recently developed microfluidic model systems that simulate the spatial microstructure of soil microbial habitats in a transparent material, which we call Soil Chips. They allow us to study the impact of soil physical microstructures on microbes, and vice versa, the influence of microbes on soil physical properties: such as Microbial behavior and interactions in response to a spatially refined habitat, or wettability and water retention, soil aggregate formation and changes in the pore space.We inoculated our chips with fluorescent lab cultures or natural whole soil inocula. Through the chips we observed via microscopy processes in real-time and at the scale of the microbial cells.We could study fungi, bacteria, protists and nematodes as well as the distribution of soil minerals and soil solution in the chips. We subjected the adjacent soil to drying-rewetting processes, which was visible in water movements inside the chip. We studied the development of preferential water flow paths, and water retention in smaller pores and as a consequence of microbial exudates. Also the microbes themselves influenced the formation of microhabitats, where fungal hyphae both blocked connections and pushed through existing borders, and single-celled protozoa opened passages through existing aggregates. We found that the presence of fungal hyphae in a pore space system increased both the presence of bacteria and the likelihood of water in the pores, and thus allowing us to study fungal highways in a more realistic soil setting.The chips act like a window into the soil, through which we can eaves-drop on a world that otherwise is largely hidden to us: Jostling protists, tsunami-like drying-rewetting events, and fungi with character. Beyond the scientific potential, the chips can also bring soils closer to people and hopefully increase engagement in soil health conservation.

Microfluidic soil chips render optical access to the naturally opaque soil systems and enable direct investigation of microbial growth and interactions in micro-structurally and chemically controlled environments. However, chemical analyses of these interactions at high spatial and temporal resolution are still lacking. Here we propose that the use of advanced microspectroscopy techniques, namely infrared absorption, Raman scattering and synchrotron radiation based X-ray microspectroscopy, in microfluidic soil chips would make it possible to approach these phenomena. They allow monitoring biogeochemical processes in and around soil microbial cells growing in the reproducibly designed microenvironments within the chips at (sub)micrometer scale. Complementary use of several of the microspectroscopy techniques is beneficial for obtaining information about both molecular and elemental composition, oxidation states and local structure of the elements in the sample. Ultimately, we argue that microspectroscopy in microfluidic chips can lead to relevant breakthroughs in frontier research areas in soil science, such as (1) analysis of chemical responses of microbes to environmental triggers at micro-scale spatial resolution, (2) phenotypical identification and phylogenetic classification of single cells of soil microbes in situ, (3) determining spatially and time resolved effects of heavy metals and organic pollutants, including microplastics, on soils and (4) spatially resolved analysis of soil organic matter dynamics for better understanding of soil carbon storage. Tailoring the chip design to achieve optical transparency to the radiation type used by the different microspectroscopy methods is crucial to achieve this; therefore, we expect that this perspective will inspire the scientific community to use the proposed approaches and thus push both the technical development of the microspectroscopy suitable soil chips and the research frontier in soil science.