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Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.

Soil microbes play an important role in governing global processes such as carbon cycling, but it is challenging to study them embedded in their natural environment and at the single cell level due to the opaque nature of the soil. Nonetheless, progress has been achieved in recent years towards visualizing microbial activities and organo-mineral interaction at the pore scale, especially thanks to the development of microfluidic 'soil chips' creating transparent soil model habitats. Image-based analyses come with new challenges as manual counting of bacteria in thousands of digital images taken from the soil chips is excessively time-consuming, while simple thresholding cannot be applied due to the background of soil minerals and debris. Here, we adopt the well-developed deep learning algorithm Mask-RCNN to quantitatively analyze the bacterial communities in soil samples from different locations in the world. This work demonstrates analysis of bacterial abundance from three contrasting locations (Greenland, Sweden and Kenya) using deep learning in microfluidic soil chips in order to characterize population and community dynamics. We additionally quantified cell- and colony morphology including cell size, shape and the cell aggregation level via calculation of the distance to the nearest neighbor. This approach allows for the first time an automated visual investigation of soil bacterial communities, and a crude biodiversity measure based on phenotypic cell morphology, which could become a valuable complement to molecular studies. • Deep learning-based image analysis recognizes microbial cells from soils in soil chips. • Cell counts were robust under contrasting acquisition in lab and field incubations. • The algorithm recognizes size, shape and spatial relationship of bacteria in communities. • The approach can be used for a morphological phenotype-based community characterization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) are considered ecosystem engineers, but the interactions of their mycelium with their immediate surroundings are largely unknown. In this study, we used microfluidic chips, simulating artificial soil structures, to study foraging strategies and habitat modification of Rhizophagus irregularis symbiotically associated to carrot roots. AMF hyphae foraged over long distances in nutrient-void spaces, preferred straight over tortuous passages, anastomosed and showed strong inducement of branching when encountering obstacles. We measured bi-directional transport of cellular content inside active hyphae and documented strategic allocation of biomass within the mycelium via cytoplasm retraction from inefficient paths. R. irregularis modified pore-spaces in the chips by clogging pores with irregularly shaped spores. We suggest that studying AMF hyphal behaviour in spatial settings can explain phenomena reported at bulk scale such as AMF modification of water retention in soils. The use of microfluidic soil chips in AMF research opens up novel opportunities to study their ecophysiology and interactions with both biotic and abiotic factors. [ABSTRACT FROM AUTHOR]

The great variety of earth's microorganisms and their functions are attributed to the heterogeneity of their habitats, but our understanding of the impact of this heterogeneity on microbes is limited at the microscale. In this study, we tested how a gradient of spatial habitat complexity in the form of fractal mazes influenced the growth, substrate degradation, and interactions of the bacterial strain Pseudomonas putida and the fungal strain Coprinopsis cinerea. These strains responded in opposite ways: complex habitats strongly reduced fungal growth but, in contrast, increased the abundance of bacteria. Fungal hyphae did not reach far into the mazes and forced bacteria to grow in deeper regions. Bacterial substrate degradation strongly increased with habitat complexity, even more than bacterial biomass, up to an optimal depth, while the most remote parts of the mazes showed both decreased biomass and substrate degradation. These results suggest an increase in enzymatic activity in confined spaces, where areas may experience enhanced microbial activity and resource use efficiency. Very remote spaces showing a slower turnover of substrates illustrate a mechanism which may contribute to the long-term storage of organic matter in soils. We demonstrate here that the sole effect of spatial microstructures affects microbial growth and substrate degradation, leading to differences in local microscale spatial availability. These differences might add up to considerable changes in nutrient cycling at the macroscale, such as contributing to soil organic carbon storage. • Bacteria and fungi respond in opposite ways to microscale physical complexity • Fungal growth decreases in complex microhabitats • Bacterial growth and nutrient degradation increase in complex habitats • Nutrients in the deepest regions of the mazes are less subject to degradation Arellano-Caicedo et al. show that bacteria and fungi react differently to complex microenvironments. Fungal growth decreases, whereas bacterial growth and nutrient degradation increase, in response to habitat complexity. These findings show the importance of considering habitat microstructure when studying microbial communities in nature. [ABSTRACT FROM AUTHOR]