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Empirical soil models reproducing soil characteristics can help to reduce the inherent complexity of soils in experiments. Microengineered or microfluidic soil chips can simulate the soil pore space at microscale in a transparent material that enables direct visual investigation of soil- and soil microbial processes including monitoring of single cells and their interactions in communities. Through the chips it is possible to control and closely monitor microhabitat conditions including oxygen levels and pH, and to single out factors such as spatial relations, pore space structure or resource patch size. They can be designed either close to realistic conditions such as based on µCT measurements, or using simple geometrical patterns that can be frequently replicated and modified within the chip design. They can thus be tailored to fit scenarios of spatially explicit soil computer models and used for iterative in-silico – in-situ experiments. We found amongst others that the geometric shape of a pore space and its connectivity influences bacterial and fungal growth, their interactions and enzymatic activity. We can measure those factors spatially resolved at cellular scale. We want to initiate a discussion for future collaborations between soil chip experimentalists and computer modelers.

Microfluidics is a multidisciplinary platform that integrates microfabrication, physical chemistry analysis, automation, and microscopy. It has the advantages of precise liquid manipulation, rapid measurements, and real-time visualization at the microscale, which is especially of interest and benefit to microbial studies. Soil Chips are microfabricated microfluidic devices typically made of glass and polydimethylsiloxane (PDMS), designed to mimic the real soil network and allow real-time visualization and characterization of microbial activity at the micro-scale. They have so far been used to investigate microbial activities, interactions, community composition, and distribution under different conditions in soil analog systems. Challenge comes when working with natural soil samples. Due to mineral aggregates and debris, valuable information such as the abundance of individuals, cell morphology, and the relationship between bacteria and their geochemical and physical environment are difficult to extract via a simple thresholding method. Since microorganisms and microfluidic structures have distinct features from the noisy background that can be easily picked up by our eyes, a biologically inspired convolutional neural network model for object detection is the most suitable tool for this task.We used a small part of data from three different experiments to train a well-developed object detection and segmentation algorithm Mask RCNN and implemented further analysis of bacteria abundance, spatial distribution, and morphological characterization. We are able to plot the distribution of all the detected bacteria including clusters in terms of abundance, size, shape, and index of aggregation. A distinct difference in bacteria characteristics can be observed in the samples acquired from three locations (Greenland, Sweden, and Kenya). We are now planning to extend the classification library to include other microbial groups including fungi, protists, invertebrates, and micro arthropods.

Micro- and Nanoplastics have become a very common pollutant of soil ecosystems, but their effect on soil microorganisms is not well understood. We exposed a model soil bacterium (Pseudomonas) and a model soil fungus (Coprinopsis) to different concentrations of nanosized polystyrene beads in microfluidic soil chips. The transparent chips allowed us to perform direct investigation of the effect of beads on abundance of the microbes and on interactions of individual cells with the nanobeads. Growth of both the bacteria and the fungi was reduced by the exposure to nanoplastics, along with a reduction in bacterial enzymatic activity. Nanobeads were strongly attracted to fungal hyphae, causing a high concentration of beads along the first hyphae to enter a pore space, and thus freeing the surrounding from a large proportion of the beads. This study contributes to our understanding of direct toxicity effects and interactions of nanoplastics to soil microbes.

Soil organisms live and interact in the intricate soil pore space labyrinth, but their natural habitat and natural interactions are difficult to study because of the opaqueness of the soil. 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, microbial behavior and realistic microbial interactions, live and at the scale of their cells.Using soil inocula, we get a large proportion of the natural microbial community into our chips and can study soil bacteria, fungi and smaller protists and nematodes in their food webs and in different spatial habitats. We were especially interested in soil protists that are generally understudied in their diversity and ecosystem functions. We were able to observe a large variety of flagellated, ciliated and amoeboid protists in the chips, predating on the bacterial populations and even on fungal hyphae. Some larger amoebae only entered the chips with their pseudopodia for predation. The colonization succession pattern of the chips showed predator-prey oscillations, with periodically high levels of different protists, followed by retreat or encystation. In chips that were containing initially dry pore spaces, colonization success of protists was strongly increased by the presence of fungal hyphae, which paved the way for protists by wetting pore spaces.The soil chips enable us to study the influence of trophic interactions such as the presence of predators on bacterial and fungal nutrient cycling. Disturbances that stronger influence protists than bacteria may have a pronounced effect on bacterial population sizes and their organic matter degradation activities. Beyond the scientific potential, the chips can also bring soils closer to people and hopefully increase engagement in soil health conservation.

A better understanding of soil carbon sequestration is important as the rapid elevation of carbon emission has significantly impacted our climate. The soil carbon dynamic process is strongly associated with trophic interactions in the soil communities. However, direct investigation of microbes and trophic interactions at microscale has been a major challenge due to the opacity of soil and the exceedingly complex pore spaces that limit direct in situ observation of trophic interactions and their variations with soil structure. As such, we decide to use microfluidic technique to create a two dimensional heterogeneous porous microenvironment containing nutrient patches of different quantity and quality to mimic the complex soil pore network and its heterogenous distribution of resources. This allows us to directly visualize and investigate the ability of organisms to access spaces starting from an initial ecophysiological precondition to changes of spatial accessibility mediated by interactions with the microbial community.Microfluidics is a multidisciplinary platform that integrates micro fabrication, physical chemistry analysis, automation and microscopy. It has been widely used in life science and chemistry as it allows precise liquid manipulation, rapid measurements, real-time visualization at microscale, which is especially of interest and benefit to microbial studies. We created a complex pore network mimicking different soil environments – earlier considered impossible to achieve experimentally. The microfluidic channel contains a random distribution of cylindrical pillars of different sizes so as to mimic the pore space variations found in real soil. The randomness in the design creates various spatial availability for microbes. The nutrient patches within the pore space are achieved via capillary force trapping or UV curing hydrogel. In the former method, the patches are created by trapping nutrients in the predesign pore space due to interfacial tension between air and nutrient liquid. However, displacement instability during air injection makes it hard to control the formation of the patches. The latter enables better and more precise patches size and location manipulation but hydrogel biocompatibility with various fungal species remains a major challenge and hydrogel curing requires special facilities. The microfluidic nutrient patches provide spatial heterogeneities of resource distribution, which allows us to have a better understanding of the influence of spatial accessibility on the microbial community. The chip has five different nutrient distribution levels, ranging from centralized – a single large nutrient patch- to increasingly dispersed nutrient distributions - sets of up to 49 loosely distributed nutrient patches of equal total volume. The experiments will be carried out using sterile cultures of fluorescent bacteria and fungi, synthetic communities of combinations of these, or a whole soil community inoculum. We will quantify the consumption of organic matter from the different areas via fluorescent substrates, and measure the bio- and necromass produced. We hypothesise that denser distribution will increase the net decomposition of organic matter as a centralized nutrient location increases the interactions within the microbial communities and individuals present in that area, which induces higher competition stress in the different communities.

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.

A grand challenge for mankind is to fight climate change, which involves both reducing and reverting CO2 emissions. Soils store more carbon (C) than the atmosphere and biosphere combined, and it is microorganisms that govern whether C compounds remain in the soil, or whether they are decomposed and released to the atmosphere as CO2. The microbial influence on C cycling range from the way they decompose soil organic matter (SOM) to their contributions on the formation of soil aggregates that are particularly important for physical C stabilization in soils. However, the relationship between the microbial activity, SOM properties and physicochemical microenvironment, including complexity of soil structure (i.e., arrangement of pore space in and between soil aggregates), and how each of these factors contribute to the prolonged residence of C in soils, is not well understood. Therefore, the aim of this work has been to develop and make use of an analytical approach for studying the influence of pore space architecture on microbial SOM decomposition and dynamics by integrating two novel tools in soil sciences – microfluidic chips, which mimic soil structure, and infrared (IR) spectroscopic imaging, which provides detailed information about chemical properties of materials within these chips.We have used several microchip designs to simulate different levels of complexity of soil pore space. The hypothesis is that the more complex the chip structures – the less decomposition of SOM will be observed, as more of it will be ‘hidden’ from its decomposers within hard-to-reach spaces. For the IR spectroscopic imaging, macro attenuated total reflection (ATR) accessory has been used. In this mode, an ATR element of high refractive index is put in contact with a sample – the microchip - and total internal reflection signal at the boundary between the element and the sample is recorded. The signal is detected with an imaging focal plane array (FPA) detector and carries information about IR absorptions in the sample. With IR spectra serving as fingerprints for identifying molecules, spatially and temporally resolved observation of chemistry and chemical changes of a SOM substrate initially filling the microchip structures and undergoing decomposition by subsequently inoculated microbial cultures can be made. Our pilot data suggests feasibility of the approach for analysis of complex substrates such as lignin, maize leaves or SOM from real soils and its dependence on the complexity of chip. Evaluating molecular changes in parts of the larger molecules or of the compound mixture under decomposition could even contribute to quantifying, e.g., N mining within the compounds. Eventually, knowing the influence of spatial structure on the decomposition rate and pathways can help us understand how important is the spatial heterogeneity when we study organic matter degradation in soils.

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.

Carbon sequestration has been a popular research topic in recent years as the rapid elevation of carbon emission has significantly impacted our climate. Apart from carbon capture and storage in e.g. oil reservoirs, soil carbon sequestration offers a long term and safe solution for the environment and human beings. The net soil carbon budget is determined by the balance between terrestrial ecosystem sink and sources of respiration to atmospheric carbon dioxide. Carbon can be long term stored as organic matters in the soil whereas it can be released from the decomposition of organic matter. The complex pore networks in the soil are believed to be able to "protect" microbial-derived organic matter from decomposition. Therefore, it is important to understand how soil structure impacts organic matter cycling at the pore scale. However, there are limited experimental studies on understanding the mechanism of physical stabilization of organic matter. Hence, my project plan is to create a heterogeneous microfluidic porous microenvironment to mimic the complex soil pore network which allows us to investigate the ability of organisms to access spaces starting from an initial ecophysiological precondition to changes of spatial accessibility mediated by interactions with the microbial community.Microfluidics is a powerful tool that enables studies of fundamental physics, rapid measurements and real-time visualisation in a complex spatial microstructure that can be designed and controlled. Many complex processes can now be visualized enabled by the development of microfluidics and photolithography, such as microbial dynamics in pore-scale soil systems and pore network modification mimicking different soil environments – earlier considered impossible to achieve experimentally. The microfluidic channel used in this project contains a random distribution of cylindrical pillars of different sizes so as to mimic the variations found in real soil. The randomness in the design creates various spatial availability for microbes (preferential flow paths with dead-end or continuous flow) as an invasion of liquids proceeds into the pore with the lowest capillary entry pressure. In order to study the impact of different porosity in isolation of varying heterogeneity of the porous medium, different pore size chips that use the same randomly generated pore network is created. Those chips have the same location of the pillars, but the relative size of each pillar is scaled. The experiments will be carried out using sterile cultures of fluorescent bacteria, fungi and protists, synthetic communities of combinations of these, or a whole soil community inoculum. We will quantify the consumption of organic matter from the different areas via fluorescent substrates, and the bio-/necromass produced. We hypothesise that lower porosity will reduce the net decomposition of organic matter as the narrower pore throat limits the access, and that net decomposition rate at the main preferential path will be higher than inside branches

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.